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Sample records for membrane-bound torpedo acetylcholine

  1. Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy.

    PubMed Central

    Zingsheim, H P; Neugebauer, D C; Frank, J; Hänicke, W; Barrantes, F J

    1982-01-01

    The acetylcholine receptor protein (AChR) from the electric organ of Torpedo marmorata is studied in its membrane-bound form by electron microscopy and single-particle image averaging. About half the molecule protrudes from the membrane surface by approximately 5 nm. The low-resolution 3-D structure of this hydrated portion, including its handedness, can be deduced from averaged axial and lateral projections and from freeze-etched membrane surfaces. In native membrane fragments, a dimeric form of the AChR is observed and the relative orientation of the AChR monomers within the dimer is established. The dimers disappear upon disulfide reduction of the membrane preparations, whereas the average axial projections of the AChR monomer remain unaffected. Since the existence of disulfide bonds linking AChR monomers between their respective delta-subunits is well documented, the approximate position of the delta-subunit within the low-resolution structure of the AChR molecule can be deduced from the structure of the dimers. Images Fig. 1. Fig. 2. Fig. 3. PMID:7188351

  2. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  3. Binding of rabies virus to purified Torpedo acetylcholine receptor.

    PubMed

    Lentz, T L; Benson, R J; Klimowicz, D; Wilson, P T; Hawrot, E

    1986-12-01

    The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

  4. In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.

    PubMed

    Ulrich, H; Ippolito, J E; Pagán, O R; Eterović, V A; Hann, R M; Shi, H; Lis, J T; Eldefrawi, M E; Hess, G P

    1998-11-24

    The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

  5. In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor

    PubMed Central

    Ulrich, Henning; Ippolito, Joseph E.; Pagán, Oné R.; Eterovic, Vesna A.; Hann, Richard M.; Shi, Hua; Lis, John T.; Eldefrawi, Mohyee E.; Hess, George P.

    1998-01-01

    The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443–473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts. PMID:9826651

  6. Development of the electromotor system of Torpedo marmorata: distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization.

    PubMed

    Richardson, G P; Fiedler, W; Fox, G Q

    1987-03-01

    A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptor-rich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electron-microscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.

  7. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  8. Effect of opioid peptides on electrically evoked acetylcholine release from Torpedo electromotor neurons.

    PubMed

    Oron, L; Sarne, Y; Michaelson, D M

    1991-04-29

    The opioid peptide dynorphin A(1-8) (1 micron) increased acetylcholine release from the Torpedo electric organ by approximately twofold. This effect was reversed by the opiate antagonist naloxone. The effect of Dyn A(1-8) on acetylcholine release was found to vary in magnitude with the seasons of the year, with maximal enhancement being observed in the summer and none in winter. Dynorphin B, methionine-enkephalin and leucine-enkephalin also increased acetylcholine release and showed similar seasonal variations. These findings suggest that acetylcholine release from Torpedo electromotor neurons is regulated by opiate receptors. The physiological significance of these observations is discussed in view of the previous findings that the Torpedo neurons contain an endogenous enkephalin-like peptide.

  9. Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

    PubMed Central

    1978-01-01

    Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti- Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha- neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state. PMID:344325

  10. Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors.

    PubMed

    Di Scala, Coralie; Baier, Carlos J; Evans, Luke S; Williamson, Philip T F; Fantini, Jacques; Barrantes, Francisco J

    2017-01-01

    Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution. © 2017 Elsevier Inc. All rights reserved.

  11. Nonopioid effect of morphine on electrically evoked acetylcholine release from Torpedo electromotor neurons.

    PubMed

    Oron, L; Sarne, Y; Michaelson, D M

    1992-02-01

    The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.

  12. Specific Stimulated Uptake of Acetylcholine by Torpedo Electric Organ Synaptic Vesicles

    NASA Astrophysics Data System (ADS)

    Parsons, Stanley M.; Koenigsberger, Robert

    1980-10-01

    The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to [14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3 about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicles were treated with nine protein modification reagents under mild conditions. Two reagents had no effect on, dithiothreitol potentiated, and six reagents strongly inhibited subsequent stimulated uptake of [3H]acetylcholine. The results indicate that uptake of acetylcholine is conditionally specific for the transported substrate, is carried out by the synaptic vesicles rather than a contaminant of the preparation, and requires a functional protein system containing a critical sulfhydryl group.

  13. Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

    PubMed Central

    Marsal, J; Tigyi, G; Miledi, R

    1995-01-01

    A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins. PMID:7761478

  14. Structural characterization of the main immunogenic region of the Torpedo acetylcholine receptor.

    PubMed

    Morell, Stuart W; Trinh, Vu B; Gudipati, Eswari; Friend, Alexander; Page, Nelson A; Agius, Mark A; Richman, David P; Fairclough, Robert H

    2014-03-01

    To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1-12), α(65-79), and α(110-115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1-12) and α(65-79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110-115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored.

  15. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    SciTech Connect

    Blanton, M.P.; Wang, H.H. )

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.

  16. Allosteric modulation of Torpedo nicotinic acetylcholine receptor ion channel activity by noncompetitive agonists.

    PubMed

    Maelicke, A; Coban, T; Storch, A; Schrattenholz, A; Pereira, E F; Albuquerque, E X

    1997-01-01

    Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.

  17. Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

    PubMed Central

    1985-01-01

    Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000- 43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending. PMID:3884630

  18. Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit.

    PubMed Central

    Claudio, T; Ballivet, M; Patrick, J; Heinemann, S

    1983-01-01

    The nucleotide sequence has been determined of a cDNA clone that codes for the 60,000-dalton gamma subunit of Torpedo californica acetylcholine receptor. The length of the cDNA clone is 2,010 base pairs. The 5' and 3' untranslated regions have respective lengths of 31 and 461 base pairs. Data suggest that the putative polyadenylylation consensus sequence A-A-T-A-A-A may not be required for polyadenylylation of the mRNA corresponding to the cDNA clone described in this study. From the DNA sequence data, the amino acid sequence of the gamma subunit was deduced. The subunit is composed of 489 amino acids giving a molecular mass of 56,600 daltons. The deduced amino acid sequence data also indicate the presence of a 17-amino acid extension or signal peptide on this subunit. From these data, structural predictions for the gamma subunit are made such as potential membrane-spanning regions, possible asparagine-linked glycosylation sites, and the assignment of regions of the protein to the extracellular, internal, and cytoplasmic domains of the lipid bilayer. Images PMID:6573658

  19. Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

    PubMed

    Eldefrawi, M E; Eldefrawi, A T; Wilson, D B

    1975-09-23

    Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.

  20. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  1. Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Parker, I; Amano, T; Miledi, R

    1984-01-01

    Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor. Images Fig. 1. PMID:6094179

  2. Aqueous Fraction of Beta vulgaris Ameliorates Hyperglycemia in Diabetic Mice due to Enhanced Glucose Stimulated Insulin Secretion, Mediated by Acetylcholine and GLP-1, and Elevated Glucose Uptake via Increased Membrane Bound GLUT4 Transporters

    PubMed Central

    Kabir, Ashraf Ul; Samad, Mehdi Bin; Ahmed, Arif; Jahan, Mohammad Rajib; Akhter, Farjana; Tasnim, Jinat; Hasan, S. M. Nageeb; Sayfe, Sania Sarker; Hannan, J. M. A.

    2015-01-01

    Background The study was designed to investigate the probable mechanisms of anti-hyperglycemic activity of B. Vulgaris. Methodology/Principal Findings Aqueous fraction of B. Vulgaris extract was the only active fraction (50mg/kg). Plasma insulin level was found to be the highest at 30 mins after B. Vulgaris administration at a dose of 200mg/kg. B. Vulgaris treated mice were also assayed for plasma Acetylcholine, Glucagon Like Peptide-1 (GLP-1), Gastric Inhibitory Peptide (GIP), Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Peptide (PACAP), Insulin Like Growth Factor-1 (IGF-1), Pancreatic Polypeptides (PP), and Somatostatin, along with the corresponding insulin levels. Plasma Acetylcholine and GLP-1 significantly increased in B. Vulgaris treated animals and were further studied. Pharmacological enhancers, inhibitors, and antagonists of Acetylcholine and GLP-1 were also administered to the test animals, and corresponding insulin levels were measured. These studies confirmed the role of acetylcholine and GLP-1 in enhanced insulin secretion (p<0.05). Principal signaling molecules were quantified in isolated mice islets for the respective pathways to elucidate their activities. Elevated concentrations of Acetylcholine and GLP-1 in B. Vulgaris treated mice were found to be sufficient to activate the respective pathways for insulin secretion (p<0.05). The amount of membrane bound GLUT1 and GLUT4 transporters were quantified and the subsequent glucose uptake and glycogen synthesis were assayed. We showed that levels of membrane bound GLUT4 transporters, glucose-6-phosphate in skeletal myocytes, activity of glycogen synthase, and level of glycogen deposited in the skeletal muscles all increased (p<0.05). Conclusion Findings of the present study clearly prove the role of Acetylcholine and GLP-1 in the Insulin secreting activity of B. Vulgaris. Increased glucose uptake in the skeletal muscles and subsequent glycogen synthesis may also play a part in

  3. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells

    PubMed Central

    1984-01-01

    The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the

  4. Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

    PubMed

    Niessen, K V; Muschik, S; Langguth, F; Rappenglück, S; Seeger, T; Thiermann, H; Worek, F

    2016-04-15

    Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100 μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ

    PubMed Central

    1981-01-01

    After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron- dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine- receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000- Mr protein binding to the inner face of the subsynaptic membrane. PMID:7287814

  6. A Raman spectroscopic investigation of the lipid state in acetylcholine receptor-rich membranes from Torpedo marmorata.

    PubMed Central

    Aslanian, D; Négrerie, M

    1985-01-01

    The lipid state in acetylcholine receptor (AcChR)-rich membranes purified from electric organ of Torpedo marmorata was studied in the temperature interval from 0 degrees C to 35 degrees C using the (C-H) stretching and (C-C) skeletal optical vibrations. The Raman spectra of AcChR-rich membranes, recorded immediately after preparation of the samples, indicate that the lipids are in a predominant triclinic crystalline lattice and do not undergo a phase transition when the temperature increases up to 35 degrees C. However, the polar groups of the lipids appear subject to temperature-induced variations. After extraction of 43-kd and other non-receptor proteins, spectra indicate an order-disorder phase transition of lipids at approximately 21 degrees C. This transition appears less cooperative than the transition of the membrane lipid extract. The role of the proteins in preservation of the crystalline state of lipids in AcChR-rich membranes is discussed. PMID:4018037

  7. Change in state of phosphorylation of acetylcholine receptor during maturation of the electromotor synapse in Torpedo marmorata electric organ.

    PubMed

    Saitoh, T; Changeux, J P

    1981-07-01

    Two populations of membrane fragments, both rich in acetylcholine receptor (AcChoR), appeared during subcellular fractionation by ultracentrifugation of neonatal Torpedo marmorata electric organs. One of these equilibrated at 38.5% (wt/wt) sucrose, as did AcChoR-rich membranes from adult fish; the other equilibrated at 36.8% sucrose. AcChoR purified from these light membrane fractions gave the same subunit profile as adult AcChoR (after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) but was more susceptible to heat inactivation and focused at an isoelectric point more alkaline by 0.1 pH unit. Treatment of adult AcChoR with Escherichia coli alkaline phosphatase decreased its thermal stability and shifted its isoelectric point towards alkaline pH. However, identical treatment did not affect AcChoR purified from neonatal light membrane fractions. The gamma and delta chains of AcChoR can be phosphorylated in vitro by endogenous protein kinases, which copurify with AcChoR-rich membranes. Treatment of AcChoR from neonatal light membranes by E. coli alkaline phosphatase enhanced the phosphorylation of the gamma and delta chains but did so to a smaller extent than in the case of adult AcChoR. In conclusion, adult AcChoR appears to be more phosphorylated than AcChoR from neonatal light membranes, indicating that its state of phosphorylation changes during development.

  8. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  9. A single gene codes for the nicotinic acetylcholine receptor alpha-subunit in Torpedo marmorata: structural and developmental implications.

    PubMed Central

    Klarsfeld, A; Devillers-Thiéry, A; Giraudat, J; Changeux, J P

    1984-01-01

    We have used Southern blot hybridization to analyze the genomic structure encoding the alpha-subunit of the acetylcholine receptor (AChR) in Torpedo marmorata, with cDNA probes isolated from the electric organ. Four different radiolabelled probes, corresponding to various parts of the alpha-subunit mRNA, hybridized to several genomic fragments of T. marmorata DNA generated by digestion with the restriction enzymes SstI, PvuII and PstI. The same hybridization pattern was observed after washing the blots under low- or high-stringency conditions. As a check for detection sensitivity of heterologous sequences, the same probes were hybridized to PvuII-digested chicken DNA, revealing bands at low stringency which disappeared at higher stringencies. Unambiguously, two of our probes (one of them entirely within the coding region) hybridized to a single genomic fragment from T. marmorata DNA. This feature, as well as the results of an extensive study of the whole hybridization pattern, points towards the uniqueness of alpha-subunit-specific sequences in the genome of T. marmorata. Since overall more bands were found than expected from the cDNA sequence, this alpha-subunit gene must be split by several introns (at least four, possibly more). The length of this gene is at least 20 kb. The existence of a single alpha-subunit gene is consistent with the absence of chemical heterogeneity in the NH2-terminal sequence of the purified alpha-chain, and supports the view that the two alpha-chains belonging to one AChR oligomer have an identical primary structure. It also suggests that localization and stabilization of the AChR in well-defined post-synaptic areas of T. marmorata electric organ basically relies, during development, on 'epigenetic' mechanisms. Images Fig. 2. Fig. 3. Fig. 4. PMID:6323168

  10. Comparison of the interactions of a specific neurotoxin (alpha-bungarotoxin) with the acetylcholine receptor in Torpedo californica and Electrophorus electricus membrane preparations.

    PubMed

    Leprince, P; Noble, R L; Hess, G P

    1981-09-15

    alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an important tool in the study of the receptor-ligand binding mechanism. Two distinct kinetic processes have been observed in studies with membranes from E. electricus. A minimum mechanism for the toxin reaction involves (i) the reversible binding of two toxin molecules to the receptor prior to the irreversible formation of toxin receptor complexes and (ii) a toxin-induced conformational change of the receptor which leads to an increase in the affinity of the receptor binding sites for toxin [Hess, G. P., Bulger, J. E., Fu, J.-j. L., Hindy, E. F., & Silberstein, R. J. (1975) Biochem. Biophys. Res. Commun. 64, 1018-1027]. Only one process has been detected in Torpedo membranes. Here, we determine whether the receptors in Torpedo californica and E. electricus membranes have different properties or whether the measurements and their interpretation were responsible for the different results. Two methods which are frequently used in binding studies to separate free and bound toxin, a CM-52 cellulose minicolumn assay and DE-81 filter disk assay, have been compared. The results obtained indicate that the interaction of toxin with receptor from T. californica is similar to that observed with receptor from E. electricus. The apparent differences which have been reported in the literature are shown to have arisen from the design of the experiments in which T. californica membranes were used.

  11. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments

    PubMed Central

    Asmar-Rovira, Guillermo A.; Asseo-García, Aloysha M.; Quesada, Orestes; Hanson, Michael A.; Nogueras, Carlos; Lasalde-Dominicci, José A.; Stevens, Raymond C.

    2009-01-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess receptor lipid composition using GC (Gas Chromatography)-FID (Flame Ionization) and/or GC-MSD (Mass Selective Detectors), stability and aggregation state using A-SEC (Analytical Size-Exclusion Chromatography) and EM (Electron Microscopy), and planar lipid bilayers to measure ion channel function. Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analog detergents [CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate), FC-12 (n-Dodecylphosphocholine) and sodium cholate (3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid)] maintaining stability and supporting ion channel function, while non-lipid analog detergents [Cymal-6 (6-Cyclohexyl-1-hexyl-β-d-maltoside), DDM (n-Dodecyl-β-d-maltopyranoside), LDAO (Lauryldimethylamine-N-oxide) and OG (n-Octyl-β-d-glucopyranoside)] showed decreased stability and significant reduction or loss of ion channel function. Anapoe-C12E9 (Polyoxyethylene-(9)-dodecyl ether) and BigCHAP (N,N′-bis-(3-d-Gluconamidopropyl) cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function when compared to lipid-analog detergents. Overall, these results show that the nAChR can be stable and functional in lipid-analog detergents or in detergents that retain moderate amounts of residual native lipids, while the opposite is true about non-lipid analog

  12. Membrane-bound selenoproteins.

    PubMed

    Liu, Jun; Rozovsky, Sharon

    2015-10-01

    Selenoproteins employ selenium to supplement the chemistry available through the common 20 amino acids. These powerful enzymes are affiliated with redox biology, often in connection with the detection, management, and signaling of oxidative stress. Among them, membrane-bound selenoproteins play prominent roles in signaling pathways, Ca(2+) regulation, membrane complexes integrity, and biosynthesis of lipophilic molecules. The number of selenoproteins whose physiological roles, protein partners, expression, evolution, and biosynthesis are characterized is steadily increasing, thus offering a more nuanced view of this specialized family. This review focuses on human membrane selenoproteins, particularly the five least characterized ones: selenoproteins I, K, N, S, and T. Membrane-bound selenoproteins are the least understood, as it is challenging to provide the membrane-like environment required for their biochemical and biophysical characterization. Hence, their studies rely mostly on biological rather than structural and biochemical assays. Another aspect that has not received much attention is the particular role that their membrane association plays in their physiological function. Findings cited in this review show that it is possible to infer the structure and the membrane-binding mode of these lesser-studied selenoproteins and design experiments to examine the role of the rare amino acid selenocysteine.

  13. Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor.

    PubMed

    Trinh, Vu B; Foster, Alex J; Fairclough, Robert H

    2014-05-01

    We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers: This 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10(-10)M, which is smaller than (7.13±1.20)×10(-10)M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10(-10)M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

  14. Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors — Recommendations for methods and experimental designs

    PubMed Central

    Losen, Mario; Martinez-Martinez, Pilar; Molenaar, Peter C.; Lazaridis, Konstantinos; Tzartos, Socrates; Brenner, Talma; Duan, Rui-Sheng; Luo, Jie; Lindstrom, Jon; Kusner, Linda

    2015-01-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR from Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice. PMID:25796590

  15. Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus: chemical kinetic measurements in the millisecond region.

    PubMed

    Hess, G P; Pasquale, E B; Walker, J W; McNamee, M G

    1982-02-01

    In earlier studies with the acetylcholine receptor (AcChoR) of Electrophorus electricus the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. Torpedo vesicles on a weight basis may contain several hundred times more receptor sites than do E. electricus vesicles. Techniques have been developed to (i) correct for the kinetic heterogeneity of the vesicle population; (ii) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted J(A), the influx rate coefficient before the onset of inactivation, to be measured); and (iii) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the E. electricus receptor accommodates the ion translocation in T. californica vesicles. The features in common are: (i) A rapid initial flux rate [J(A)(max) for T. californica is 310 sec(-1); for E. electricus it is 7.5 sec(-1)]. These differences in flux rates are consistent with a difference in AcChoR density. (ii) A rapid inactivation process [alpha(max) for T. californica is 2 sec(-1); for E. electricus it is 7 sec(-1)]. (iii) A slow AcChoR-controlled flux that continues after the rapid inactivation [J(I)(max) for T. californica is 1.3 sec(-1); for E. electricus it is 0.015 sec(-1)]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, beta, of 0.12 sec(-1). The second

  16. Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus: Chemical kinetic measurements in the millisecond region

    PubMed Central

    Hess, George P.; Pasquale, Elena B.; Walker, Jeffrey W.; McNamee, Mark G.

    1982-01-01

    In earlier studies with the acetylcholine receptor (AcChoR) of Electrophorus electricus the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. Torpedo vesicles on a weight basis may contain several hundred times more receptor sites than do E. electricus vesicles. Techniques have been developed to (i) correct for the kinetic heterogeneity of the vesicle population; (ii) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted JA, the influx rate coefficient before the onset of inactivation, to be measured); and (iii) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the E. electricus receptor accommodates the ion translocation in T. californica vesicles. The features in common are: (i) A rapid initial flux rate [JA(max) for T. californica is 310 sec-1; for E. electricus it is 7.5 sec-1]. These differences in flux rates are consistent with a difference in AcChoR density. (ii) A rapid inactivation process [α(max) for T. californica is 2 sec-1; for E. electricus it is 7 sec-1]. (iii) A slow AcChoR-controlled flux that continues after the rapid inactivation [JI(max) for T. californica is 1.3 sec-1; for E. electricus it is 0.015 sec-1]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, β, of 0.12 sec-1. The second process leads to undetectable

  17. Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analog [125I]-SADU-3-72

    PubMed Central

    Pandhare, Akash; Hamouda, Ayman K.; Staggs, Brandon; Aggarwal, Shaili; Duddempudi, Phaneendra K.; Lever, John R.; Lapinsky, David J.; Jansen, Michaela; Cohen, Jonathan B.; Blanton, Michael P.

    2012-01-01

    Bupropion, a clinically-used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analog, (±)-2-(N-tert-butylamino)-3′-[125I]-iodo-4′-azidopropiophenone (SADU-3-72). Based upon inhibition of [125I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with three-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [125I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [125I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu265, βLeu257) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP, but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket. PMID:22394379

  18. Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor

    PubMed Central

    Cruz-Martín, A.; Mercado, J.L.; Rojas, L.V.; McNamee, M.G.; Lasalde-Dominicci, J.A.

    2015-01-01

    Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC50, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC50s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. PMID:11547353

  19. Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor.

    PubMed

    Padilla-Morales, Luis F; Morales-Pérez, Claudio L; De La Cruz-Rivera, Pamela C; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A; Quesada, Orestes; Lasalde-Dominicci, José A

    2011-10-01

    Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β(2)-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

  20. Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor

    PubMed Central

    Padilla-Morales, Luis F.; Morales-Pérez, Claudio L.; De La Cruz-Rivera, Pamela C.; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A.

    2011-01-01

    Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility. PMID:21922299

  1. Use of chemical modifications and site-directed mutagenesis to probe the functional role of thiol groups on the. gamma. subunit of Torpedo californica acetylcholine receptor

    SciTech Connect

    Pradier, L.; Yee, A.S.; McNamee, M.G. )

    1989-08-08

    Alkylation of Torpedo californica purified nicotinic acetylcholine receptor (AChR) with N-phenylmaleimide (NPM) under nonreducing conditions led to ion flux inhibition without affecting ligand binding properties. The {gamma} subunit was shown to be preferentially labeled by ({sup 3}H)NPM with partial labeling of the {alpha} subunit at higher NPM concentrations. Alkylation occurs at cysteine residues as confirmed by amino acid analysis. Cyanogen bromide peptide mapping of the {gamma} subunit indicates that at least two residues corresponding to Cys-416, -420, or -451 are labeled. Residues 416 and 420 are part of the proposed amphipathic helix, and the functional role of these two cysteines is further investigated by site-directed mutagenesis of T. californica AChR cDNAs and expression of the mutants in Xenopus laevis oocytes following injection of SP6 transcripts. Several features of SP6 transcripts are shown to be important for efficient translation in vivo. Mutations Cys {yields} Ser{gamma}416,420 and Cys {yields} Phe{gamma}416 did not perturb either the receptor functional properties or its expression levels. The double mutant Cys {yields} Phe{gamma}416,420 displayed a 30% decrease of normalized AChR activity. The relatively small effect of large steric mutations in the amphipathic helix argues against its presence in the tightly packed transmembrane domain of the protein.

  2. Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte

    PubMed Central

    1989-01-01

    The assembly of the nicotinic acetylcholine receptor (AchR) and the 43- kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR- rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings. PMID:2642909

  3. Torpedo Rockets

    NASA Technical Reports Server (NTRS)

    2004-01-01

    All through the 13th to the 15th Centuries there were reports of many rocket experiments. For example, Joanes de Fontana of Italy designed a surface-rurning, rocket-powered torpedo for setting enemy ships on fire

  4. Torpedo Rockets

    NASA Technical Reports Server (NTRS)

    2004-01-01

    All through the 13th to the 15th Centuries there were reports of many rocket experiments. For example, Joanes de Fontana of Italy designed a surface-rurning, rocket-powered torpedo for setting enemy ships on fire

  5. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane

    PubMed Central

    1979-01-01

    The receptor-rich postsynaptic membrane of the elasmobranch electric organ was fixed by quick-freezing and then viewed by freeze-fracture, deep-etching and rotary-replication. Traditional freeze-fracture revealed a distinct, geometrical pattern of shallow 8.5-nm bumps on the E fracture-face, similar to the lattice which has been seen before in chemically fixed material, but seen less clearly than after quick- freezing. Fracture plus deep-etching brought into view on the true outside of this membrane a similar geometrical pattern of 8.5-nm projections rising out of the membrane surface. The individual projections looked like structures that have been seen in negatively stained or deep-etched membrane fragments and have been identified as individual acetylcholine receptor molecules. The surface protrusions were twice as abundant as the large intramembrane particles that characterize the fracture faces of this membrane, which have also been considered to be receptor molecules. Particle counts have always been too low to match the estimates of postsynaptic receptor density derived from physiological and biochemical studies; counts of surface projections, however, more closely matched these estimates. Rotary- replication of quick-frozen, etched postsynaptic membranes enhanced the visibility of these surface protuberances and illustrated that they often occur in dimers, tetramers, and ordered rows. The variations in these surface patterns suggested that in vivo, receptors in the postsynaptic membrane may tend to pack into "liquid crystals" which constantly appear, flow, and disappear in the fluid environment of the membrane. Additionally, deep-etching revealed a distinct web of cytoplasmic filaments beneath the postsynaptic membrane, and revealed the basal lamina above it; and delineated possible points of contact between these structures and the membrane proper. PMID:479296

  6. Chemical kinetic measurements of the effect of trans- and cis-3,3'-Bis[(trimethylammonio)methyl]azobenzene bromide on acetylcholine receptor mediated ion translocation in Electrophorus electricus and Torpedo californica.

    PubMed

    Delcour, A H; Hess, G P

    1986-04-08

    A quench-flow technique was used to study the effect of trans- and cis-3,3'-bis[(trimethylammonio)methyl]azobenzene bromide (trans- and cis-Bis-Q), photoisomerizable ligands, on the acetylcholine receptor in vesicles prepared from the electric organ of Electrophorus electricus and of Torpedo californica. In E. electricus, two rate coefficients of the receptor-mediated translocation of 86Rb+ induced with trans-Bis-Q were measured: JA, the rate coefficient for ion flux, and alpha, the rate coefficient for receptor inactivation (desensitization). Both rate coefficients increase with increasing concentrations of Bis-Q up to 50 microM. At higher concentrations JA decreases in a concentration-dependent manner while alpha remains unchanged. This effect was previously observed with suberyldicholine [Pasquale, E. B., Takeyasu, K., Udgaonkar, J., Cash, D.J., Severski, M.C., & Hess, G. P. (1983) Biochemistry 22, 5967-5973] and with acetylcholine [Takeyasu, K., Udgaonkar, J., & Hess, G. P. (1983) Biochemistry 22, 5973-5978] and was analyzed in terms of a minimum mechanism that accounts for the properties of activation, desensitization, and inhibition of the receptor. Two molecules of trans-Bis-Q must be bound for the channel to open, but at concentrations greater than 50 microM the population of open channels decreases because of the additional binding of one molecule of trans-Bis-Q to a regulatory inhibitory site, independent of the activating sites. cis-Bis-Q does not induce transmembrane ion flux, but it does inhibit the response of the receptor to acetylcholine and induces inactivation (desensitization) in the micromolar range.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. 73. TORPEDO WORK SHOP FORWARD LOOKING AFT SHOWING TORPEDO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    73. TORPEDO WORK SHOP - FORWARD LOOKING AFT SHOWING TORPEDO ELEVATOR, HIGH PRESSURE AIR REDUCING STATION, SCUTTLEBUTT, TORPEDO AFTERBODY WORKSHOP, OVERHEAD CHAIN MOIST AND RAIL SYSTEM AND OVERHEAD SPRINKLER SYSTEM. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  8. Conformational phases of membrane bound cytoskeletal filaments

    NASA Astrophysics Data System (ADS)

    Quint, David A.; Grason, Gregory; Gopinathan, Ajay

    2013-03-01

    Membrane bound cytoskeletal filaments found in living cells are employed to carry out many types of activities including cellular division, rigidity and transport. When these biopolymers are bound to a membrane surface they may take on highly non-trivial conformations as compared to when they are not bound. This leads to the natural question; What are the important interactions which drive these polymers to particular conformations when they are bound to a surface? Assuming that there are binding domains along the polymer which follow a periodic helical structure set by the natural monomeric handedness, these bound conformations must arise from the interplay of the intrinsic monomeric helicity and membrane binding. To probe this question, we study a continuous model of an elastic filament with intrinsic helicity and map out the conformational phases of this filament for various mechanical and structural parameters in our model, such as elastic stiffness and intrinsic twist of the filament. Our model allows us to gain insight into the possible mechanisms which drive real biopolymers such as actin and tubulin in eukaryotes and their prokaryotic cousins MreB and FtsZ to take on their functional conformations within living cells.

  9. Structure Biology of Membrane Bound Enzymes

    SciTech Connect

    Fu, Dax

    2016-11-30

    The overall goal of the proposed research is to understand the membrane-associated active processes catalyzed by an alkane $\\square$-hydroxylase (AlkB) from eubacterium Pseudomonase oleovorans. AlkB performs oxygenation of unactivated hydrocarbons found in crude oils. The enzymatic reaction involves energy-demanding steps in the membrane with the uses of structurally unknown metal active sites featuring a diiron [FeFe] center. At present, a critical barrier to understanding the membrane-associated reaction mechanism is the lack of structural information. The structural biology efforts have been challenged by technical difficulties commonly encountered in crystallization and structural determination of membrane proteins. The specific aims of the current budget cycle are to crystalize AlkB and initiate X-ray analysis to set the stage for structural determination. The long-term goals of our structural biology efforts are to provide an atomic description of AlkB structure, and to uncover the mechanisms of selective modification of hydrocarbons. The structural information will help elucidating how the unactivated C-H bonds of saturated hydrocarbons are oxidized to initiate biodegradation and biotransformation processes. The knowledge gained will be fundamental to biotechnological applications to biofuel transformation of non-edible oil feedstock. Renewable biodiesel is a promising energy carry that can be used to reduce fossil fuel dependency. The proposed research capitalizes on prior BES-supported efforts on over-expression and purification of AlkB to explore the inner workings of a bioenergy-relevant membrane-bound enzyme.

  10. Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

    PubMed Central

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

    2014-01-01

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

  11. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    PubMed

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-04

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  12. The "torpedo" effect in medicine.

    PubMed

    Tsoucalas, Gregory; Karamanou, Marianna; Lymperi, Maria; Gennimata, Vassiliki; Androutsos, George

    2014-01-01

    The natural electrical phenomena fascinated humans since antiquity. The electrical discharges produced by the torpedo fish were highly appreciated among ancient physicians as Hippocrates, Scribonius Largus and Galen and were prescribed for headache, gout and prolapsed anus. In the medieval period, torpedo's electrical properties were attributed to occult powers, while Renaissance physicians' and scientists' studied the anatomy and mechanical nature of the provoked shock paving the way for the discovery of the electrical nature of torpedo's activity and the evolution of electrotherapy.

  13. Torpedo electromotor system development: biochemical differentiation of Torpedo electrocytes in vitro.

    PubMed

    Richardson, G P; Witzemann, V

    1986-04-01

    The accumulation of 2 postsynaptic proteins--the acetylcholine receptor and acetylcholinesterase, total protein and lactate dehydrogenase levels, and the evolution of the multiple molecular forms of acetylcholinesterase (exhibiting apparent sedimentation coefficients of 17, 13, 11 and 6S) have been examined in aneural cultures of embryonic Torpedo electric organ explanted before, during or after electrocyte differentiation and the onset of synaptogenesis. During electrocyte differentiation in vitro, with explants taken before the 38 mm stage, the relative proportions of the 17, 13 and 11S forms change in vitro as in vivo but the 6S form remains abnormally dominant. In tissue explants taken from 38 to 47 mm stage embryos, the 4 major molecular forms of acetylcholinesterase differentiate in a manner identical to that observed in vivo. In explants taken after the onset of synaptogenesis (55-80 mm stages), the proportions of the acetylcholinesterase forms change as in vivo only during the first week in vitro whilst accumulation is occurring at the normal in vivo rate. The switch to the high acetylcholine receptor and acetylcholinesterase accumulation rate that occurs when synaptogenesis begins in vivo is not observed after any time lag in vitro with tissue explanted before the stage (55 mm) at which synaptogenesis begins. The effects on acetylcholinesterase and acetylcholine receptor accumulation of supplementing the medium with a neural tissue extract are described. The experiments were designed to elucidate the factors and mechanisms that regulate the differentiation and formation of chemical synapses using the electric organ of Torpedo marmorata as a model system. The results demonstrate that the complex changes occurring in the multiple molecular forms of acetylcholinesterase during electrocyte differentiation are not under direct neural control but that the switch to an increased acetylcholinesterase and acetylcholine receptor accumulation rate may be triggered by

  14. Membrane-bound mucin modular domains: from structure to function.

    PubMed

    Jonckheere, Nicolas; Skrypek, Nicolas; Frénois, Frédéric; Van Seuningen, Isabelle

    2013-06-01

    Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.

  15. Staining membrane-bound proteins with coomassie blue r250.

    PubMed

    Stochaj, Wayne R; Berkelman, Tom; Laird, Nancy

    2006-10-01

    INTRODUCTIONCoomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 μg of protein. One drawback of Coomassie Blue staining is that it produces a high background that can make interpretation of results difficult.

  16. Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

    PubMed

    Jay, D; Jay, E G; Garcia, C

    1993-12-01

    Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

  17. Tunable Tensor Voting Improves Grouping of Membrane-Bound Macromolecules

    SciTech Connect

    Loss, Leandro A.; Bebis, George; Parvin, Bahram

    2009-04-15

    Membrane-bound macromolecules are responsible for structural support and mediation of cell-cell adhesion in tissues. Quantitative analysis of these macromolecules provides morphological indices for damage or loss of tissue, for example as a result of exogenous stimuli. From an optical point of view, a membrane signal may have nonuniform intensity around the cell boundary, be punctate or diffused, and may even be perceptual at certain locations along the boundary. In this paper, a method for the detection and grouping of punctate, diffuse curvilinear signals is proposed. Our work builds upon the tensor voting and the iterative voting frameworks to propose an efficient method to detect and refine perceptually interesting curvilinear structures in images. The novelty of our method lies on the idea of iteratively tuning the tensor voting fields, which allows the concentration of the votes only over areas of interest. We validate the utility of our system with synthetic and annotated real data. The effectiveness of the tunable tensor voting is demonstrated on complex phenotypic signals that are representative of membrane-bound macromolecular structures.

  18. Photochemical energy conversion by membrane-bound photoredox systems

    SciTech Connect

    Tollin, G.

    1992-03-01

    Most of our effort during the past grant period has been directed towards investigating electron transfer processes involving redox proteins at lipid bilayer/aqueous interfaces. This theme, as was noted in our previous three year renewal proposal, is consistent with our goal of developing biomimetic solar energy conversion systems which utilize the unique properties of biological electron transfer molecules. Thus, small redox proteins such as cytochrome c, plastocyanin and ferredoxin function is biological photosynthesis as mediators of electron flow between the photochemical systems localized in the membrane, and more complex soluble or membrane-bound redox proteins which are designed to carry out specific biological tasks such as transbilayer proton gradient formation, dinitrogen fixation, ATP synthesis, dihydrogen synthesis, generation of strong reductants, etc. In these studies, we have utilized two principal experimental techniques, laser flash photolysis and cyclic voltammetry, both of which permit direct measurements of electron transfer processes.

  19. Reverse signaling through membrane-bound interleukin-15.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Pohl, Thomas; Borden, Ernest C; Silverman, Robert; Bulfone-Paus, Silvia

    2004-10-01

    The results from this study implicate membrane-anchored interleukin (IL)-15 constitutively expressed on the cell surface of PC-3 human prostate carcinoma cells and interferon-gamma-activated human monocytes in reverse signaling upon stimulation with soluble IL-15 receptor-alpha or anti-IL-15 antibodies, mediating the outside-to-inside signal transduction that involves the activation of members of the MAPK family (ERK and p38) and focal adhesion kinase. The presence of membrane-bound IL-15 was not dependent on the expression of the trimeric IL-15 receptor complex by these cells and resisted treatment with acidic buffer or trypsin. Reverse signaling through membrane-bound IL-15 considerably increased the production of several pro-inflammatory cytokines by monocytes, such as IL-6, IL-8, and tumor necrosis factor-alpha, thereby indicating the relevance of this process to the complex immunomodulatory function of these cells. Furthermore, stimulation of transmembrane IL-15 also enhanced the transcription of IL-6 and IL-8 in the PC-3 cell line and promoted migration of PC-3 cells as well as LNCaP human prostate carcinoma cells stably expressing IL-15 on the cell surface. Thus, IL-15 can exist as a biologically active transmembrane molecule that possesses dual ligand-receptor qualities with a potential to induce bidirectional signaling. This fact highlights a new level of complexity in the biology of IL-15 and offers novel important insights into our understanding of the cellular responses modulated by this pleiotropic cytokine.

  20. Soluble low-Km 5'-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage.

    PubMed

    Vogel, M; Kowalewski, H; Zimmermann, H; Hooper, N M; Turner, A J

    1992-06-15

    Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

  1. Cytochemical identification of the leukocytes of torpedoes (Torpedo marmorata Risso and Torpedo ocellata Rafinisque).

    PubMed

    Grimaldi, M C; D'lppolito, S; Pica, A; Della Corte, F

    1983-01-01

    The authors subjected peripheral blood smears of Torpedoes to cytochemical analysis of lipids, protein, neutral and acid polysaccahrides and of some enzymatic activities, i.e. adenosine triphosphatase (ATP-ase), acid and alkaline phosphatase, aliesterase and peroxidase. It was found that neutrophilic granulocytes are intensely PAS and aliesterase positive and weakly ATP-ase positive. Eosinophilic granulocytes show the presence of neutral polysaccharides in the matrix (which is PAS positive) and strong ATP-ase and acid phosphatase activities in the granules. Lymphocytes sometimes contain weakly PAS and aliesterase positive granules. Monocytes show some small PAS positive granules and weak acid phosphatase and aliesterase activities. Thrombocytes contain some peripheral granules which are PAS positive and slightly ATP-ase positive. There are no transitional forms between the various cellular types. The results confirm the classification of leukocytes of Torpedoes into neutrophilic granulocytes, eosinophilic granulocytes, lymphocytes, monocytes and thrombocytes and contribute some informations about the histoenzymatic content of Elasmobranch leukocytes.

  2. [The study of role of membrane-bound Ca2+ in the regulation of relationship between neuron and glia during rhythmic excitation].

    PubMed

    Maksimov, G V; Turovetskiĭ, V B; Chaterjy, Ch; Andreev, A I; Mironova, Iu E; Brindikova, T A; Rubin, A B

    2000-01-01

    The role of membrane-bound Ca2+ in the regulation of Ca2+ transport through voltage-gated Ca2+ channel, and NMDA-glutamate and n-acetylcholine receptors upon interaction of a neuron with glia during rhythmic excitation was studied. It was found that the redistribution and transport of Ca2+ play a crucial role in the conductance of rhythmic excitation in both a "neuron-neuron" system and the processes providing the maintenance of a stationary level of rhythmic excitation in the system "neuron-glia".

  3. Membrane-bound beta-lactamase forms in Escherichia coli.

    PubMed

    Plückthun, A; Pfitzinger, I

    1988-10-05

    Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site. These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane. The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth. We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane. Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se. If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane. If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm. If only the first signal contains a cleavage site, a longer fusion protein is transported and released. The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species.

  4. Heterologous expression and purification of membrane-bound pyrophosphatases.

    PubMed

    Kellosalo, J; Kajander, T; Palmgren, M G; Lopéz-Marqués, R L; Goldman, A

    2011-09-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.

  5. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  6. Mark XIV Torpedo Case Study

    DTIC Science & Technology

    2011-02-26

    to cry and the XO and I said ‘Captain, this ship was tracking right on course with the speed and course we got it exactly right.’ He said ‘We will...they realized how much testing they really needed to do. The inherent arrogance of the Gun Club and the “not invented here” syndrome , have been...The silent service—The torpedoes of WW II ( Cat # AAE 43107) [History Channel documentary]. New York, NY: A&E Television Networks. Naval Undersea

  7. The electromotor system of Torpedo. A model cholinergic system.

    PubMed

    Whittaker, V P

    1977-12-01

    The electric organ of Torpedo, besides providing abundant amounts of cholinoceptive post-synaptic membrane for the isolation of the acetylcholine receptor protein, is a rich source of cholinergic nerve terminals. Using perfused, innervated tissue blocks from which synaptic vesicles in different functional states can be isolated, much information can be obtained about synaptic-vesicle dynamics. So far this is consistent with the view that the synaptic vesicles are the source of transmitter released on stimulation and that uptake of newly synthesized transmitter by the vesicles is dependent on their having discharged their previous charge of transmitter in at least one cycle of exo- and endocytosis. Studies of the protein composition of the vesicle membrane, especially when combined with similar information about the external presynaptic membrane, purified samples of which are now available from synaptosome (T-sac) preparations, promise to throw new light on the molecular mechanism underlying vesicle exo-/endocytosis.

  8. Meprins, membrane-bound and secreted astacin metalloproteinases

    PubMed Central

    Sterchi, Erwin E.; Stöcker, Walter; Bond, Judith S.

    2008-01-01

    The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the ‘hatching’ subfamily comprising alveolin, ovastacin, LCE, HCE (‘low’ and ‘high’ choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions. PMID:18783725

  9. Action of suramin upon ecto-apyrase activity and synaptic depression of Torpedo electric organ.

    PubMed Central

    Martí, E.; Cantí, C.; Gómez de Aranda, I.; Miralles, F.; Solsona, C.

    1996-01-01

    1. The role of ATP, which is co-released with acetylcholine in synaptic contacts of Torpedo electric organ, was investigated by use of suramin. Suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulphoni c acid], a P2 purinoceptor antagonist, potently inhibited in a non-competitive manner the ecto-apyrase activity associated with plasma membrane isolated from cholinergic nerve terminals of Torpedo electric organ. The Ki was 30 microM and 43 microM for Ca(2+)-ADPase and Ca(2+)-ATPase respectively. 2. In Torpedo electric organ, repetitive stimulation decreased the evoked synaptic current by 51%. However, when fragments of electric organ were incubated with suramin the evoked synaptic current declined by only 14%. Fragments incubated with the selective A1 purinoceptor antagonist, DPCPX, showed 5% synaptic depression. 3. The effects of suramin and DPCPX on synaptic depression were not addictive. Synaptic depression may thus be linked to endogenous adenosine formed by dephosphorylation of released ATP by an ecto-apyrase. The final effector in synaptic depression, adenosine, acts via the A1 purinoceptor. 4. ATP hydrolysis is prevented in the presence of suramin. It slightly increased (20%) the mean amplitude of spontaneous miniature endplate currents. The frequency distribution of the amplitude of spontaneous events was shifted to the right, indicating that ATP, when not degraded, may modulate the activation of nicotinic acetylcholine receptors activated by the quantal secretion of acetycholine. PMID:8818348

  10. 11. Submersible torpedo tube mounted on platform of elevator at ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Submersible torpedo tube mounted on platform of elevator at northeast (starboard) elevator tower. - Naval Torpedo Station, Firing Pier, North end of Gould Island in Narragansett Bay, Newport, Newport County, RI

  11. The noncompetitive blocker ( sup 3 H)chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: Implications for the alpha-helical organization of regions MII and for the structure of the ion channel

    SciTech Connect

    Revah, F.; Galzi, J.L.; Giraudat, J.; Haumont, P.Y.; Lederer, F.; Changeux, J.P. )

    1990-06-01

    Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker ({sup 3}H)chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by ({sup 3}H)chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The ({sup 3}H)chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.

  12. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: (/sup 3/H)chlorpromazine labels homologous residues in the. beta. and delta chains

    SciTech Connect

    Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P.Y.; Lederer, F.; Changeux, J.P.

    1987-05-05

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker (/sup 3/H)chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled ..beta.. chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by (/sup 3/H)chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the ..beta.. chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

  13. Delayed luminescence of luminol initiated by a membrane-bound peroxidase.

    PubMed

    Ikariyama, Y; Suzuki, S; Aizawa, M

    1981-09-01

    The luminescense of the luminol-H2O2 system was initiated by either free or membrane-bound horseradish peroxideae (HRP). The instantaneous luminescene decayed rapidly and was followed by the delayed luminescence in the presence of excess luminol. The delayed luminescence was characterized by a chain reaction, in which luminescence intensity increased exponentially. Membrane-bound HRP demonstrated that the delayed luminescence took place even in the absence of HRP if the instantaneous luminescence was initiated by HRP. A mechanism for the nonenzymatic luminescence is proposed and discussed.

  14. Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants.

    PubMed

    Lüthje, Sabine; Meisrimler, Claudia-Nicole; Hopff, David; Möller, Benjamin

    2011-07-01

    Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.

  15. Configuration of membrane-bound proteins by x-ray reflectivity

    NASA Astrophysics Data System (ADS)

    Chen, Chiu-Hao; Málková, Šárka; Cho, Wonhwa; Schlossman, Mark L.

    2011-11-01

    In this presentation we review our recent work using x-ray reflectivity to determine the configuration of membrane-bound proteins. The reflectivity data is analyzed in terms of the known crystallographic structure of proteins and a slab model representing the lipid layer to yield an electron density profile of the lipid/protein system. Our recent modified analysis methodology for the lipid/protein system is concisely described in this report. In addition, some results of the configuration of the membrane-bound proteins cPLA2α-C2, p40phox-PX, and PKCα-C2 are highlighted.

  16. AN IMPROVED CELL FRACTIONATION PROCEDURE FOR THE PREPARATION OF RAT LIVER MEMBRANE-BOUND RIBOSOMES

    PubMed Central

    Adelman, M. R.; Blobel, Gunter; Sabatini, David D.

    1973-01-01

    A cell fractionation procedure is described which allows the preparation from rat liver of a rough microsome population containing almost 50% of the membrane-bound ribosomes of the tissue. The fraction is not contaminated with free ribosomes or smooth microsomes, and, by various other criteria, is suitable for studies of ribosome-membrane interaction. PMID:4345164

  17. Copper(II) enhances membrane-bound α-synuclein helix formation.

    PubMed

    Lucas, Heather R; Lee, Jennifer C

    2011-03-01

    Interactions of copper and membranes with α-synuclein have been implicated in pathogenic mechanisms of Parkinson's disease, yet work examining both concurrently is scarce. We have examined the effect of copper(ii) on protein/vesicle binding and found that both the copper(ii) affinity and α-helical content are enhanced for the membrane-bound protein.

  18. NMR studies of the membrane bound form of filamentous bacteriophage fd and Pfl major coat proteins

    SciTech Connect

    Schiksnis, R.A.; Bogusky, M.J.; Opella, S.J.

    1987-05-01

    The major coat proteins of the fd (M13) and Pf1 filamentous bacteriophage exist as integral membrane proteins during the viral life cycle. These proteins adopt their membrane bound conformations when solubilized by a variety of detergents, and the protein-detergent micelle complexes can be studied using solution NMR techniques. Determination of the structure of the coat proteins in their membrane bound form has been accomplished by qualitative interpretation of 2-dimensional /sup 1/H-/sup 1/H NOE spectra (NOESY). The critical amide proton resonance assignments were made through biosynthetic /sup 15/N labeling and /sup 1/H//sup 15/N heteronuclear chemical shift correlation techniques. The data indicate that both proteins adopt helical conformations within the micelle. The /sup 15/N//sup 1/H heteronuclear NOE has been used to characterize the backbone dynamics of both proteins in micelles. The lipid associated residues of the proteins are rigid on the nanosecond timescale, while the hydrophilic solvent associated N- and C-termini are high mobile. These results complement previously reported protein dynamics studies of membrane bound coat proteins conducted using solid state NMR methods. Solid state NMR studies reported in the literature have also investigated the structure and dynamics of the fd and Pf1 major coat proteins when bound to intact phage. Therefore, structure/dynamics comparisons of the proteins in their structural versus membrane bound forms can be made.

  19. Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

    PubMed

    Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

    2017-10-01

    Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l(-1) and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. On the chimerical nature of the membrane-bound ATPase from halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1991-01-01

    A series of experiments are described that were carried out with the goal of determining how the membrane-bound ATPase from H. saccharovorum is related to V- and F-type ATPases. They reflect three approaches: the use of inhibitors; structural studies; and immunological relatedness.

  1. Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

    PubMed Central

    2011-01-01

    Background During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. PMID:21798097

  2. Structural Study of Purkinje Cell Axonal Torpedoes in Essential Tremor

    PubMed Central

    Louis, Elan D.; Yi, Hong; Erickson-Davis, Cordelia; Vonsattel, Jean-Paul G.; Faust, Phyllis L.

    2009-01-01

    Background Essential tremor (ET) is one of the most common neurological diseases. A basic understanding of its neuropathology is now emerging. Aside from Purkinje cell loss, a prominent finding is an abundance of torpedoes (rounded swellings of Purkinje cell axons). Such swellings often result from the mis-accumulation of cell constituents. Identifying the basic nature of these accumulations is an important step in understanding the underlying disease process. Torpedoes, only recently identified in ET, have not yet been characterized ultrastructurally. Objectives Light and electron microscopy were used to characterize the structural constituents of torpedoes in ET. Methods Formalin-fixed cerebellar cortical tissue from 4 prospectively-collected ET brains was sectioned and immunostained with a monoclonal phosphorylated neurofilament antibody (SMI-31, Covance, Emeryville, CA). Using additional sections from 3 ET brains, torpedoes were assessed using electron microscopy. Results Immunoreactivity for phosphorylated neurofilament protein revealed clear labeling of torpedoes in each case. Torpedoes were strongly immunoreactive; in many instances, two or more torpedoes were noted in close proximity to one another. On electron microscopy, torpedoes were packed with randomly arranged 10–12 nm neurofilaments. Mitochondria and smooth endoplasmic reticulum were abundant as well, particularly at the periphery of the torpedo. Conclusions We demonstrated that the torpedoes in ET represent the mis-accumulation of disorganized neurofilaments and other organelles. It is not known where in the pathogenic cascade these accumulations occur (i.e., whether these accumulations are the primary event or a secondary/downstream event) and this deserves further study. PMID:19047012

  3. Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

    PubMed

    Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

    2016-04-19

    Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

  4. Torpedoes and Their Impact on Naval Warfare

    DTIC Science & Technology

    2017-01-01

    This history of the torpedo traces the weapon’s evolution from Robert Whitehead’s invention of a self-propelled underwater missile in 1866 to the...not the intent of this book to glorify a weapon that has caused such immense suffering and destruction but rather to trace the torpedo’s evolution and...cut off. This book attempts to present a rational, unbiased record of the torpedo’s evolution and to document the significant impact that this

  5. Detection of membrane-bound and soluble antigens by magnetic levitation.

    PubMed

    Andersen, Mikkel Schou; Howard, Emily; Lu, Shulin; Richard, Matthew; Gregory, Mark; Ogembo, Gordon; Mazor, Ofer; Gorelik, Pavel; Shapiro, Nathan I; Sharda, Anish V; Ghiran, Ionita

    2017-09-14

    Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.

  6. From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein.

    PubMed

    Vos, Werner L; Nazarov, Petr V; Koehorst, Rob B M; Spruijt, Ruud B; Hemminga, Marcus A

    2009-05-01

    The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.

  7. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

  8. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    PubMed

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  9. Using supported bilayers to study the spatiotemporal organization of membrane bound proteins

    PubMed Central

    Field, Christine M.; Groen, Aaron C.; Mitchison, Timothy J.

    2015-01-01

    Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence (TIRF) microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking and cell motility. PMID:25997350

  10. [Membrane-bound proteases involved in neuropeptide degradation in the brain].

    PubMed

    Yokosawa, H

    1993-07-01

    The action of neuropeptides at the synapse is terminated through enzymatic degradation by membrane-bound proteases. We defined and purified membrane-bound proteases functioning at the initial stage of degradation of four neuropeptides. 1. Substance P-degrading endopeptidases isolated from the rat brain and pig striatum showed similar properties to those of endopeptidase-24.16 (neurolysin) except for cleavage sites of substance P. 2. LHRH fragment (1-5)-generating endopeptidases isolated from the neuroblastoma cells and rat brain showed similar properties to those of endopeptidase-24.15 (thimet oligopeptidase). 3. One of two dynorphin-degrading cysteine proteases isolated from neuroblastoma cells showed strict specificity toward the Arg-Arg residues. 4. Endopeptidase-24.11 (neprilysin) isolated from the rat brain was identified as a somatostatin-degrading enzyme.

  11. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

    PubMed

    Mishra, Suman; Mishra, Rajnikant

    2016-05-01

    The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.

  12. Synthetic activity enhancement of membrane-bound lipase from Rhizopus chinensis by pretreatment with isooctane.

    PubMed

    Wang, Dong; Xu, Yan; Teng, Yun

    2007-05-01

    The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4 degrees C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.

  13. Compartmentalized system with membrane-bound glycerol kinase. Activity and product distribution versus asymmetrical substrate supply.

    PubMed Central

    Girard, A; Merchie, B; Maïsterrena, B

    1991-01-01

    An artificial-membrane-bound glycerokinase chosen as a membrane-bound two-substrate-enzyme model has been used to separate two unequal compartments of a specially designed diffusion cell. An interesting feature is the asymmetry of compartments and the existence of a diffusion layer adjacent to only one face of the enzymic membrane. In such a situation the apparent enzyme activity and the product distribution in the system have been studied versus all the possibilities of combination of ATP and glycerol supply. Our approach has lead us to differentiate two different roles played by a diffusion layer adjacent to a permeable enzymic membrane. Depending on the spatial origin of the enzymic substrates (i.e. from which compartment they derive), the diffusion layer can play either the role of a passive additional resistance to that of the membrane or the role of a third compartment in which the reaction product can partially accumulate before splitting on both parts of the membrane. Our results mainly demonstrate that a membrane-bound enzyme activity and the resulting product distribution occurring in a compartmentalized system may be regulated by the cumulative effect due to the asymmetry in volumes of the compartments, the presence of a diffusion layer and the different possibilities of substrate supply. With the topography studied, which is close to that reported for many 'in vivo' situations, the product may be diffused lead to vectorial metabolism processes. PMID:2012608

  14. The catalytic function of cytochrome P450 is entwined with its membrane-bound nature.

    PubMed

    Barnaba, Carlo; Gentry, Katherine; Sumangala, Nirupama; Ramamoorthy, Ayyalusamy

    2017-01-01

    Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

  15. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.; Wade, Rebecca C.

    2011-08-11

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

  16. Membrane bound IL-15 is increased on CD14 monocytes in early stages of MS.

    PubMed

    Vaknin-Dembinsky, Adi; Brass, Steven D; Brass, Steven; Gandhi, Roopali; Weiner, Howard L

    2008-03-01

    IL-15 is a pro-inflammatory cytokine whose three-dimensional structure is similar to that of IL-2. IL-2 and IL-15 have similar as well as distinct biological functions. An active form of IL-15 that is membrane bound has also been described. Furthermore, IL-15 is known to play a role in autoimmune diseases. We thus investigated the expression of membrane bound IL-15 on monocytes (CD14+ cells) and studied its effect on T cell activation in MS patients. We found that unstimulated CD14+ cells from relapsing remitting MS patients had increased membrane bound IL-15. Those with high surface levels of IL-15 on monocytes were in the early stages of the disease. In addition, we found that T cells of MS patients had enhanced responsiveness to IL-15 and there was increased expression of IL-15 receptor on CD4+ T cells. Thus, IL-15 may be an important cytokine that drives Th1 responses early in the course of the disease and could serve as a target for immunotherapy and as an early marker in the immunologic staging of MS.

  17. Distinguishing torpedo maculopathy from similar lesions of the posterior segment.

    PubMed

    Villegas, Victor M; Schwartz, Stephen G; Flynn, Harry W; Capó, Hilda; Berrocal, Audina M; Murray, Timothy G; Harbour, J William

    2014-01-01

    Torpedo maculopathy is a congenital solitary, oval-shaped lesion typically located temporal to the center of the macula. Congenital hypertrophy of the retinal pigment epithelium (RPE), RPE lesions of Gardner syndrome, and other lesions can present with similar characteristics. Because of its unique clinical and imaging features, torpedo maculopathy generally can be differentiated from other posterior segment lesions.

  18. Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

    SciTech Connect

    Belkin, V.M.; Volodarskaya, S.M.

    1986-06-20

    The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes.

  19. Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues.

    PubMed Central

    Chattopadhyay, A; Mukherjee, S; Rukmini, R; Rawat, S S; Sudha, S

    1997-01-01

    The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes. PMID:9251800

  20. Coordination of copper to the membrane-bound form of α-synuclein.

    PubMed

    Dudzik, Christopher G; Walter, Eric D; Abrams, Benjamin S; Jurica, Melissa S; Millhauser, Glenn L

    2013-01-08

    Aggregation of the 140-amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal-dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane-bound forms. Previous work evaluated the Cu(2+) uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the N-terminus of the protein and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate the coordination of copper to the membrane-bound form of α-syn. In this molecular environment, Cu(2+) binds exclusively to the N-terminus of the protein (Met1-Asp2) with no participation from His50. Copper does not alter the membrane-bound α-syn conformation or enhance the release of the protein from the bilayer. The Cu(2+) affinity is similar to that identified for solution α-syn, suggesting that copper coordination is retained in the membrane. Consideration of these results demonstrates that copper exerts its greatest conformational effect on the solution form of α-syn.

  1. Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

    PubMed

    Simão, Ana Maria S; Beloti, Márcio M; Cezarino, Rodrigo M; Rosa, Adalberto Luiz; Pizauro, João M; Ciancaglini, Pietro

    2007-04-01

    Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

  2. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  3. Coordination of Copper to the Membrane-Bound Form of α-Synuclein

    SciTech Connect

    Dudzik, Christopher G.; Walter, Eric D.; Abrams, Benjamin S.; Jurica, Melissa S.; Millhauser, Glenn L.

    2013-01-01

    Aggregation of the 140 amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane bound forms. Previous work evaluated the Cu2+ uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the protein N-terminus and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate copper coordination to the membrane bound form of α-syn. In this molecular environment, Cu2+ binds exclusively to the protein N-terminus (Met1-Asp2) with no participation from His50. Copper does not alter the membrane bound α-syn conformation, or enhance the protein's release from the bilayer. The Cu2+ affinity is similar to that identified for solution α-syn suggesting that copper coordination is retained in the membrane. Consideration of these results suggests that copper exerts its greatest conformational affect on the solution form of α-syn and this species may therefore be precursor to PD arising from environmental copper exposure.

  4. Membrane-bound transcription factors: regulated release by RIP or RUP.

    PubMed

    Hoppe, T; Rape, M; Jentsch, S

    2001-06-01

    Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

  5. Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Soluble Nanolipoprotein Particles

    SciTech Connect

    Baker, S E; Hopkins, R C; Blanchette, C; Walsworth, V; Sumbad, R; Fischer, N; Kuhn, E; Coleman, M; Chromy, B; Letant, S; Hoeprich, P; Adams, M W; Henderson, P T

    2008-10-22

    Hydrogenases constitute a promising class of enzymes for ex vivo hydrogen production. Implementation of such applications is currently hindered by oxygen sensitivity and, in the case of membrane-bound hydrogenases (MBH), poor water solubility. Nanolipoprotein particles (NLPs), formed from apolipoproteins and phospholipids, offer a novel means to incorporate MBH into in a well-defined water-soluble matrix that maintains the enzymatic activity and is amenable to incorporation into more complex architectures. We report the synthesis, hydrogen-evolving activity and physical characterization of the first MBH-NLP assembly. This may ultimately lead to the development of biomimetic hydrogen production devices.

  6. Study of the interaction of cadmium with membrane-bound succinate dehydrogenase.

    PubMed

    Jay, D; Zamorano, R; Muñoz, E; Gleason, R; Boldu, J L

    1991-04-01

    Cadmium ions inhibit membrane-bound succinate dehydrogenase with a second-order rate constant of 10.42 mM-1 s-1 at pH 7.35 and 25 degrees C. Succinate and malonate protect the enzyme against cadmium ion inhibition. The protection pattern exerted by succinate and malonate suggests that the group modified by cadmium is located at the active site. The pH curve of inactivation by Cd2+ indicates the involvement of an amino acid residue with pKa of 7.23.

  7. Label-free detection of clustering of membrane-bound proteins.

    PubMed

    Carton, Ixaskun; Brisson, Alain R; Richter, Ralf P

    2010-11-15

    We report a new and label-free method to detect and characterize the clustering of membrane-bound proteins and, by extension, the lateral segregation of nanosized particles adsorbed to planar surfaces in liquid environment. The method exploits the contrast between two different mass and surface sensitive detection methods, quartz crystal microbalance with dissipation monitoring and ellipsometry. The time-resolved correlation of both techniques provides insight into subtle changes in the clustering state of surface-bound molecules that is not accessible with either technique alone. A theoretical model can provide quantitative predictions about the size of surface-bound clusters.

  8. Torpedo: topic periodicity discovery from text data

    NASA Astrophysics Data System (ADS)

    Wang, Jingjing; Deng, Hongbo; Han, Jiawei

    2015-05-01

    Although history may not repeat itself, many human activities are inherently periodic, recurring daily, weekly, monthly, yearly or following some other periods. Such recurring activities may not repeat the same set of keywords, but they do share similar topics. Thus it is interesting to mine topic periodicity from text data instead of just looking at the temporal behavior of a single keyword/phrase. Some previous preliminary studies in this direction prespecify a periodic temporal template for each topic. In this paper, we remove this restriction and propose a simple yet effective framework Torpedo to mine periodic/recurrent patterns from text, such as news articles, search query logs, research papers, and web blogs. We first transform text data into topic-specific time series by a time dependent topic modeling module, where each of the time series characterizes the temporal behavior of a topic. Then we use time series techniques to detect periodicity. Hence we both obtain a clear view of how topics distribute over time and enable the automatic discovery of periods that are inherent in each topic. Theoretical and experimental analyses demonstrate the advantage of Torpedo over existing work.

  9. Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

    SciTech Connect

    Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu )

    1990-01-09

    Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

  10. Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set.

    PubMed

    Chang, Hang; Wen, Quan; Parvin, Bahram

    2015-03-01

    Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance.

  11. SEC-MALLS ANALYSIS OF HYALURONAN SIZE DISTRIBUTIONS MADE BY MEMBRANE-BOUND HYALURONAN SYNTHASE

    PubMed Central

    Baggenstoss, Bruce A.; Weigel, Paul H.

    2006-01-01

    SEC-MALLS analyses of E. coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10–100 MDa) that co-eluted with HA, and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65–75°C eliminated this artifact and increased the yield of recovered HA, due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed UDP produced during the reaction, improved the linearity of HA synthesis - even at high substrate utilization. Surprisingly, addition of EDTA, to chelate Mg+2 ions, did not completely stop the HAS reaction at 30°C or at 4°C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4–6 MDa within 2–4 hours. PMID:16476403

  12. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    PubMed

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

  13. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    PubMed

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.

  14. Interaction of the membrane-bound succinate dehydrogenase with substrate and competitive inhibitors.

    PubMed

    Kotlyar, A B; Vinogradov, A D

    1984-01-18

    The protective effect of dicarboxylates on the active-site-directed inhibition of the membrane-bound succinate dehydrogenase by N-ethylmaleimide, steady-state kinetics methods for Ki and Ks determinations, and equilibrium studies were employed to quantitate the relative affinities of succinate, fumarate, malonate and oxaloacetate to the reduced and oxidized species of the enzyme. A more than 10-fold difference in the relative affinities of the reduced and oxidized succinate dehydrogenase to succinate, fumarate and oxaloacetate is found, whereas the reactivity of the active-site sulphydryl group does not depend on the redox state of the enzyme. The redox-state-dependent changes in the affinity of the membrane-bound succinate dehydrogenase to oxaloacetate can be quantitatively accounted for by a 10-fold increase in the rate of dissociation of the enzyme-inhibitor complex which occurs upon reduction of the enzyme. The data obtained give no support for either the existence of a sulphydryl group other than the active-site one important for the catalysis or for the presence of a separate dicarboxylate-specific regulatory site in the succinate dehydrogenase molecule.

  15. Purification and characterization of a membrane-bound sialidase from pig liver.

    PubMed

    Kobayashi, T; Ito, M; Ikeda, K; Tanaka, K; Saito, M

    2000-04-01

    A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.

  16. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis.

    PubMed

    Sakamoto, Kanta; Van Veen, H W; Saito, Hiromi; Kobayashi, Hiroshi; Konings, Wil N

    2002-11-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 microM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N'-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the alpha- and beta-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.

  17. Membrane-bound α-synuclein interacts with glucocerebrosidase and inhibits enzyme activity

    PubMed Central

    Yap, Thai Leong; Velayati, Arash; Sidransky, Ellen; Lee, Jennifer C.

    2012-01-01

    Mutations in GBA, the gene encoding glucocerebrosidase, the lysosomal enzyme deficient in Gaucher disease increase the risk for developing Parkinson disease. Recent research suggests a relationship between glucocerebrosidase and the Parkinson disease-related amyloid-forming protein, α-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. Previously, we showed that α-synuclein and glucocerebrosidase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of α-synuclein by either promoting protein degradation and/or preventing aggregation. Here, we demonstrate that membrane-bound α-synuclein interacts with glucocerebrosidase, and that this complex formation inhibits enzyme function. Using site-specific fluorescence and Förster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the glucocerebrosidase-α-synuclein interaction involves a larger α-synuclein region compared to that found in solution. In addition, α-synuclein acts as a mixed inhibitor with an apparent IC50 in the submicromolar range. Importantly, the membrane-bound, α-helical form of α-synuclein is necessary for inhibition. This glucocerebrosidase interaction and inhibition likely contribute to the mechanism underlying GBA-associated parkinsonism. PMID:23266198

  18. Membrane-Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis

    PubMed Central

    Sakamoto, Kanta; van Veen, H. W.; Saito, Hiromi; Kobayashi, Hiroshi; Konings, Wil N.

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N′-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the α- and β-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds. PMID:12406727

  19. Nucleotide specificity for the bidirectional transport of membrane-bounded organelles in isolated axoplasm.

    PubMed

    Leopold, P L; Snyder, R; Bloom, G S; Brady, S T

    1990-01-01

    Video microscopy of isolated axoplasm from the squid giant axon permits correlated quantitative analyses of membrane-bounded organelle transport both in the intact axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane-bounded organelles can be evaluated under nearly physiological conditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of axoplasm with 2-20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to less than 50% of optimal values when the axoplasm was perfused with 10-20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP-gamma-S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were revealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well-defined in vitro assays to develop models for the molecular mechanisms of axonal transport.

  20. Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley

    SciTech Connect

    Klimczak, L.J.; Hind, G. )

    1990-04-01

    The soluble and membrane-bound forms of the calcium-dependent protein kinase from barley leaves (Hordeum vulgare L. cv. Borsoy) have been partially purified and compared. Both forms showed an active polypeptide of 37 kilodaltons on activity gels with incorporated histone as substrate. They eluted from chromatofocusing columns at an identical isoelectric point of pH 4.25 {plus minus} 0.2, and also comigrated on several other chromatographic affinity media including Matrex Gel Blue A, histone-agarose, phenyl-Sepharose, and heparin-agarose. Both activities comigrated with chicken ovalbumin during gel filtration through Sephacryl S-200, indicating a native molecular mass of 45 kilodaltons. The activities share a number of enzymatic properties including salt and pH dependence, free calcium stimulation profile, substrate specificity, and Km values. The soluble activity was shown to bind to artificial lipid vesicles. These data suggest strongly that the soluble and membrane-bound calcium-dependent protein kinases from barley are very closely related or even identical.

  1. Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set

    PubMed Central

    Wen, Quan

    2014-01-01

    Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance. PMID:25530633

  2. Study on torpedo fuze signal denoising method based on WPT

    NASA Astrophysics Data System (ADS)

    Zhao, Jun; Sun, Changcun; Zhang, Tao; Ren, Zhiliang

    2013-07-01

    Torpedo fuze signal denoising is an important action to ensure reliable operation of fuze. Based on the good characteristics of wavelet packet transform (WPT) in signal denoising, the paper used wavelet packet transform to denoise the fuze signal under a complex background interference, and a simulation of the denoising results with Matlab is performed. Simulation result shows that the WPT denoising method can effectively eliminate background noise exist in torpedo fuze target signal with higher precision and less distortion, leading to advance the reliability of torpedo fuze operation.

  3. The Development of Primary Cell Batteries for Torpedoes and Midget Submarine in Germany (Die Entwicklung von Primaerelementbatterien fuer Torpedos und Kleinst-U-Boote in Deutschland),

    DTIC Science & Technology

    1982-10-05

    displacement 1314 1 Water capacity 141 1 Negative buoyancy 20.4 % A run of 9000 meters was calculated at a speed of 30 kn. Torpedo G 76 - II ( Gerat 20... future , owing to the critical war situation the work on this torpedo was discontinued by decision of the Torpedo Committee at the TVA, as well as at IG...longer compatible with the requirements of antisubmarine warfare, and that in the future thermal motive power in torpedoes will take on ever greater

  4. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

  5. Pacific Coast Torpedo Station, Keyport Industrial District, Both sides of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Pacific Coast Torpedo Station, Keyport Industrial District, Both sides of Second Street, between Dedrick Drive and Liberty Bay and one building west of Dedrick Drive and south of Second Street, Keyport, Kitsap County, WA

  6. Evaluation of Protective Coatings for Aluminum Torpedoes. Phase 2.

    DTIC Science & Technology

    1988-04-01

    48 is primarily made of 6000 and 7000 series aluminum , both of which are lightweight and strong, but have poor -~ resistance to corrosion. On the...D-A195 153 EVALUATION OF PROTECTIVE COATINGS FOR ALUMINUM 1 TORPEDOES PHASE 20U) CONSTRUCTION ENGINEERING RESEARCH LAB (ARMY) CHAMPAIGN IL S R...Engineering Research Laboratory AD-A195 153 Evaluation of Protective Coatings for Aluminum Torpedoes: Phase II by Susan A. Johnston This report

  7. Corrections to the Saffman-Delbrück Mobility for Membrane Bound Proteins

    PubMed Central

    Naji, Ali; Levine, Alex J.; Pincus, P. A.

    2007-01-01

    Recent experiments (Gambin, Y., R. Lopez-Esparza, M. Reffay, E. Sierecki, N. S. Gov, M. Genest, R. S. Hodes, and W. Urbach. 2006. Proc. Natl. Acad. Sci. USA. 103:2098–2102) have called into question the applicability of the Saffman-Delbrück diffusivity for proteins embedded in the lipid bilayers. We present a simple argument to account for this observation that should be generically valid for a large class of transmembrane and membrane bound proteins. Whenever the protein-lipid interactions locally deform the membrane, that deformation generates new hydrodynamic stresses on the protein-membrane complex leading to a suppression of its mobility. We show that this suppression depends on the protein size in a manner consistent with the work of Gambin et al. PMID:17872958

  8. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    NASA Astrophysics Data System (ADS)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  9. The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Kristjansson, Hordur; Altekar, Wijaya

    1987-01-01

    The procedure for the isolation and 70-fold purification of membrane-bound cold-sensitive ATPase from Halobacterium saccharovorum is described. Upon exposure to cold, the enzyme dissociates into two major subunits, I (87 kDa) and II (60 kDa), and two minor subunits, III (29 kDa) and IV (20 kDa). The stoichiometry of the enzyme is proposed to be I2.II2.III.IV; the molecular mass of such a complex would be 343 kDa, which is in good agreement with the value of 350 kDa obtained by gel filtration. The structure of the ATPase from H. saccharovorum makes it unlike any previously described ATPase.

  10. Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

    PubMed Central

    Flanagan, Lindsey A.; Parkin, Alison

    2016-01-01

    Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

  11. Corrections to the Saffman-Delbruck mobility for membrane bound proteins.

    PubMed

    Naji, Ali; Levine, Alex J; Pincus, P A

    2007-12-01

    Recent experiments (Gambin, Y., R. Lopez-Esparza, M. Reffay, E. Sierecki, N. S. Gov, M. Genest, R. S. Hodes, and W. Urbach. 2006. Proc. Natl. Acad. Sci. USA. 103:2098-2102) have called into question the applicability of the Saffman-Delbrück diffusivity for proteins embedded in the lipid bilayers. We present a simple argument to account for this observation that should be generically valid for a large class of transmembrane and membrane bound proteins. Whenever the protein-lipid interactions locally deform the membrane, that deformation generates new hydrodynamic stresses on the protein-membrane complex leading to a suppression of its mobility. We show that this suppression depends on the protein size in a manner consistent with the work of Gambin et al.

  12. The purification and subunit structure of a membrane-bound ATPase from the Archaebacterium Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Kristjansson, Hordur; Altekar, Wijaya

    1987-01-01

    The procedure for the isolation and 70-fold purification of membrane-bound cold-sensitive ATPase from Halobacterium saccharovorum is described. Upon exposure to cold, the enzyme dissociates into two major subunits, I (87 kDa) and II (60 kDa), and two minor subunits, III (29 kDa) and IV (20 kDa). The stoichiometry of the enzyme is proposed to be I2.II2.III.IV; the molecular mass of such a complex would be 343 kDa, which is in good agreement with the value of 350 kDa obtained by gel filtration. The structure of the ATPase from H. saccharovorum makes it unlike any previously described ATPase.

  13. Purification and characterization of the membrane-bound nitrate reductase isoenzymes of Bradyrhizobium japonicum.

    PubMed

    Fernández-López, M; Olivares, J; Bedmar, E J

    1996-08-19

    Two respiratory membrane-bound nitrate reductase (NR) isoenzymes, NRI and NRII, have been purified for the first time from one single microorganism. Triton X-100-solubilized NRs were purified by a three-step procedure of differential centrifugation, Q-Sepharose chromatography, and gel filtration on Sephacryl S-300. Both isoenzymes were purified to homogeneity by the criteria of NR activity staining in polyacrylamide gels run under non-denaturating conditions and coincident staining of the protein band by silver nitrate. NRI is composed of three subunits of 116 kDa, 68 kDa, and 56 kDa, whereas NRII is composed of four subunits of 116 kDa, 68 kDa, 59 kDa, and 56 kDa. The 116-kDa subunit of NRI and the 59-kDa subunit of NRII exhibited immunological cross-reactivity with the respiratory NR of Pseudomonas stutzeri strain ZoBell.

  14. Genome-Based Discovery of a Novel Membrane-Bound 1,6-Dihydroxyphenazine Prenyltransferase from a Marine Actinomycete

    PubMed Central

    Zeyhle, Philipp; Bauer, Judith S.; Kalinowski, Jörn; Shin-ya, Kazuo; Gross, Harald; Heide, Lutz

    2014-01-01

    Recently, novel prenylated derivatives of 1,6-dihydroxyphenazine have been isolated from the marine sponge-associated Streptomyces sp. SpC080624SC-11. Genome sequencing of this strain now revealed a gene cluster containing all genes necessary for the synthesis of the phenazine and the isoprenoid moieties. Unexpectedly, however, the cluster did not contain a gene with similarity to previously investigated phenazine prenyltransferases, but instead a gene with modest similarity to the membrane-bound prenyltransferases of ubiquinone and menaquinone biosynthesis. Expression of this gene in E. coli and isolation of the membrane fraction proved that the encoded enzyme, Mpz10, catalyzes two successive prenylations of 1,6-dihydroxyphenazine. Mpz10 is the first example of a membrane-bound enzyme catalyzing the prenylation of a phenazine substrate, and one of few examples of membrane-bound enzymes involved in the prenylation of aromatic secondary metabolites in microorganisms. PMID:24892559

  15. Effect of aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes.

    PubMed

    Rajasekaran, S; Sriram, N; Arulselvan, P; Subramanian, S

    2007-03-01

    Diabetes mellitus is known to promote deterioration of membrane function and impair intra cellular metabolism in the organism. The aim of the present study was to examine the effect of the ethanolic extract from Aloe vera leaf gel on membrane bound phosphatases and lysosomal hydrolases in the liver and kidney of streptozotocin (STZ)-induced diabetic rats. The rats treated with STZ showed significant alterations in the activities of membrane bound phosphatases and lysosomal hydrolases in the liver and kidney. Oral administration of Aloe vera gel extract at a dose of 300 mg/kg body weight/day to STZ-induced diabetic rats for a period of 21 days significantly restored the alterations in enzymes activity to near normalcy. These results were compared with glibenclamide, a reference drug. Thus, the present study confirms that Aloe vera gel extract possesses a significant beneficial effect on membrane bound phosphatases and lysosomal hydrolases.

  16. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

    PubMed Central

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  17. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    SciTech Connect

    Baumgold, J.

    1986-05-01

    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  18. Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

    PubMed

    Sampedro, Javier; Valdivia, Elene R; Fraga, Patricia; Iglesias, Natalia; Revilla, Gloria; Zarra, Ignacio

    2017-02-01

    In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer.

  19. Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

    PubMed Central

    Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

    2014-01-01

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

  20. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.

  1. Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.

    PubMed

    Koshy, Seena S; Eyles, Stephen J; Weis, Robert M; Thompson, Lynmarie K

    2013-12-10

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

  2. Proinflammatory cytokines and their membrane-bound receptors are altered in the lymphocytes of schizophrenia patients.

    PubMed

    Pandey, Ghanshyam N; Ren, Xinguo; Rizavi, Hooriyah S; Zhang, Hui

    2015-05-01

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in the plasma/serum of schizophrenia (SZ) patients. To examine if SZ is also associated with the abnormal gene expression of cytokines and their membrane-bound receptors, we studied mRNA expression of proinflammatory cytokines and their receptors in lymphocytes of SZ patients and normal control (NC) subjects. We determined the protein and mRNA expression of proinflammatory cytokines and mRNA expression of their receptors in lymphocytes from 30 SZ patients and 30 drug-free NC subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mRNA levels of IL-6, TNF-α, IL-1R1, TNFR1, and TNFR2, but not IL-1β, IL-1R2, IL-1RA, IL-6R, or GP130 were significantly increased in lymphocytes of SZ patients compared with NC subjects. We also found that the protein expression of IL-6 and TNF-α, but not IL-1β, was also significantly increased in SZ patients compared with NC subjects. These studies suggest that in addition to the reported abnormalities of proinflammatory cytokines and their soluble receptors in the plasma of SZ patients, an abnormal gene expression of these cytokines and their membrane-bound receptors may be involved in the pathogenesis of SZ.

  3. Purification of acetylcholine receptors from the muscle of Electrophorus electricus.

    PubMed

    Lindstrom, J M; Cooper, J F; Swanson, L W

    1983-08-02

    Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.

  4. The pharmacology of novel acetylcholinesterase inhibitors, (+/-)-huprines Y and X, on the Torpedo electric organ.

    PubMed

    Ros, E; Aleu, J; Gómez de Aranda, I; Muñoz-Torrero, D; Camps, P; Badia, A; Marsal, J; Solsona, C

    2001-06-08

    The effects of the tacrine-huperzine A hybrid acetylcholinesterase inhibitors, (+/-)-12-amino-3-chloro-9-methyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride ((+/-)-huprine Y) and (+/-)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride ((+/-)-huprine X), were tested on spontaneous synaptic activity by measuring the amplitude, the rise time, the rate of rise, the half-width and the area or the electrical charge of the miniature endplate potentials (m.e.p.ps) recorded extracellularly on Torpedo electric organ fragments. (+/-)-Huprine Y and (+/-)-huprine X at a concentration of 500 nM increased all the m.e.p.p. variables analyzed. The effect of (+/-)-huprine Y was smaller than that of (+/-)-huprine X for all the variables except for the rate of rise where there was no significant difference. The effects of these drugs were also tested on nicotinic receptors by analyzing the currents elicited by acetylcholine (100 microM) in Xenopus laevis oocytes, transplanted with membranes from Torpedo electric organ. Both drugs inhibited the currents in a reversible manner, (+/-)-huprine Y (IC(50)=452 nM) being more effective than (+/-)-huprine X (IC(50)=4865 nM). The Hill coefficient was 0.5 for both drugs. The inhibition of the nicotinic receptor was voltage-dependent and decreased at depolarizing potentials, and there was no significant difference in the effects between (+/-)-huprine Y and (+/-)-huprine X at concentrations near to their IC(50) values. At depolarizing potentials between -20 and +15 mV, these drugs did not have any detectable effect on the blockade of the nicotinic receptor. Both huprines increased the desensitization of the nicotinic receptors since the current closed quickly in the presence of the drugs, and there was no significant difference in this effect between (+/-)-huprine Y (500 nM) and (+/-)-huprine X (5 microM). We conclude that (+/-)-huprine Y and (+/-)-huprine X increase the level of

  5. Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate.

    PubMed

    Miras-Portugal, M T; Millaruelo, A; Vara, F

    1980-12-10

    Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

  6. 44. 'Submarine Torpedo Tube Foundation and Towers,' Y&D Drawing 226855, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    44. 'Submarine Torpedo Tube Foundation and Towers,' Y&D Drawing 226855, approved by Bureau of Ordnance. Dated 20 October 1943. - Naval Torpedo Station, Firing Pier, North end of Gould Island in Narragansett Bay, Newport, Newport County, RI

  7. Irreversible thermal denaturation of Torpedo californica acetylcholinesterase.

    PubMed Central

    Kreimer, D. I.; Shnyrov, V. L.; Villar, E.; Silman, I.; Weiner, L.

    1995-01-01

    Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state. PMID:8563632

  8. Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors

    PubMed Central

    Natale, Christopher A; Duperret, Elizabeth K; Zhang, Junqian; Sadeghi, Rochelle; Dahal, Ankit; O'Brien, Kevin Tyler; Cookson, Rosa; Winkler, Jeffrey D; Ridky, Todd W

    2016-01-01

    The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. DOI: http://dx.doi.org/10.7554/eLife.15104.001 PMID:27115344

  9. Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

    SciTech Connect

    Stevens, B.R.; Kempner, E.S.; Wright, E.M.

    1986-11-01

    gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.

  10. A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Kortlüke, C; Horstmann, K; Schwartz, E; Rohde, M; Binsack, R; Friedrich, B

    1992-01-01

    One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units. Images PMID:1383192

  11. Inhibition of membrane bound ATPases of Escherichia coli and Listeria monocytogenes by plant oil aromatics.

    PubMed

    Gill, A O; Holley, R A

    2006-09-01

    Previous studies have reported that the mechanism of bactericidal action of the plant oil aromatics, eugenol, carvacrol and cinnamaldehyde involves inhibition of adenosine triphosphate generation and membrane disruption. In this study the capacity of the aromatics to inhibit the membrane bound ATPase activity of Escherichia coli and Listeria monocytogenes was investigated by experiments on isolated membranes. Inhibition of the ATPase activity of E. coli membranes was observed with 5 mM or 10 mM eugenol or carvacrol. Progressively greater inhibition by cinnamaldehyde was observed as concentration increased from 0.1 to 10 mM. L. monocytogenes ATPase activity was significantly inhibited by eugenol (5 or 10 mM), carvacrol (10 mM) and cinnamaldehyde (10 mM). Lactobacillus sakei is highly resistant to cinnamaldehyde compared to E. coli and L. monocytogenes. To determine whether this resistance was related to the relative hydrophobicity of the cell surface and hence the ability of the cell to take up the aromatics, the percentage of the three organisms partitioning in dodecane was compared. No significant difference was found between the partitioning percentage of L. monocytogenes (17.2%) and L. sakei (13.8%), indicating that surface hydrophobicity does not explain the differing sensitivity to cinnamaldehyde of these two organism. The percent partitioning of E. coli was significantly greater than both other organisms (23.3%) and may explain the greater sensitivity of E. coli to all three aromatics.

  12. Ligand-modulated conformational switching in a fully synthetic membrane-bound receptor

    NASA Astrophysics Data System (ADS)

    Lister, Francis G. A.; Le Bailly, Bryden A. F.; Webb, Simon J.; Clayden, Jonathan

    2017-05-01

    Signal transduction through G-protein-coupled receptors (GPCRs) involves binding to signalling molecules at the cell surface, which leads to global changes in molecular conformation that are communicated through the membrane. Artificial mechanisms for communication involving ligand binding and global conformational switching have been demonstrated so far only in the solution phase. Here, we report a membrane-bound synthetic receptor that responds to binding of a ligand by undergoing a conformational change that is propagated over several nanometres, deep into the phospholipid bilayer. Our design uses a helical foldamer core, with structural features borrowed from a class of membrane-active fungal antibiotics, ligated to a water-compatible, metal-centred binding site and a conformationally responsive fluorophore. Using the fluorophore as a remote reporter of conformational change, we find that binding of specific carboxylate ligands to a Cu(II) cofactor at the binding site perturbs the foldamer's global conformation, mimicking the conformational response of a GPCR to ligand binding.

  13. [Interaction of surface-active base with fraction of membrane-bound Williams's protons].

    PubMed

    Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

    2013-01-01

    In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

  14. Recent advances in plant membrane-bound transcription factor research: emphasis on intracellular movement.

    PubMed

    Seo, Pil Joon

    2014-04-01

    Transcription factors constitute numerous signal transduction networks and play a central role in gene expression regulation. Recent studies have shown that a limited portion of transcription factors are anchored in the cellular membrane, storing as dormant forms. Upon exposure to environmental and developmental cues, these transcription factors are released from the membrane and translocated to the nucleus, where they regulate associated target genes. As this process skips both transcriptional and translational regulations, it guarantees prompt response to external and internal signals. Membrane-bound transcription factors (MTFs) undergo several unique steps that are not involved in the action of canonical nuclear transcription factors: proteolytic processing and intracellular movement. Recently, alternative splicing has also emerged as a mechanism to liberate MTFs from the cellular membranes, establishing an additional activation scheme independent of proteolytic processing. Multiple layers of MTF regulation add complexity to transcriptional regulatory scheme and ensure elaborate action of MTFs. In this review, we provide an overview of recent findings on MTFs in plants and highlight the molecular mechanisms underlying MTF liberation from cellular membranes with an emphasis on intracellular movement. © 2013 Institute of Botany, Chinese Academy of Sciences.

  15. Role of nickel in membrane-bound hydrogenase and nickel metabolism in Rhizobium japonicum

    SciTech Connect

    Stults, L.W.

    1986-01-01

    The membrane-bound hydrogenase of Rhizobium japonicum requires nickel for activity. Radioactive /sup 63/Ni co-migrates with hydrogenase activity in native gel systems and co-elutes with purified hydrogenase form an affinity matrix column. A simplified scheme for the purification of hydrogenase has been developed and constitutes the first report of the aerobic purification of this enzyme from R. japonicum. The aerobic purification utilizes the general affinity matrix. Reactive Red 120-agarose and results in higher specific activity and yield of enzyme than previously reported. The stability of aerobically purified hydrogenase to oxygen is substantially greater than that reported for anaerobically isolated enzyme. Reduction of the aerobically purified enzyme in the presence of oxygen, however, results in the rapid loss of activity. R. japonicum cells accumulate nickel during heterotrophic growth and as non-growing cells. The hydrogenase constitutive mutant SR470 accumulates substantially greater amounts of nickel under both conditions. Kinetic studies indicate that the nickel uptake system in the hydrogenase constitutive mutant SR470 is upregulated relative to SRwt cells. The uptake system is specific for nickel, although a 10-fold excess (relative to nickel) of copper or zinc inhibits nickel uptake. The nickel uptake system appears to require energy. Under nickel-free conditions hydrogenase protein is not synthesized as determined by cross-reactivity with antibodies directed against hydrogenase, indicating that nickel regulates the formation of the enzyme as well as being a constituent of the active protein.

  16. A Dynamic Model of Membrane-Bound Phospholipase Cβ2 Activation by Gβγ Subunits

    PubMed Central

    Han, Daniel S.; Golebiewska, Urszula; Stolzenberg, Sebastian; Weinstein, Harel

    2011-01-01

    Phospholipase C (PLC) β2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into secondary messengers, can be activated by the Gβγ subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLCβ2 mediates both the response to Gβγ and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLCβ2 in its activated form complexed with Gβγ and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLCβ2-PH domain specificity for Gβγ and lipid membranes and suggest a mechanism for the previously reported dependence of Gβγ activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes. PMID:21693623

  17. Purification and structural analysis of membrane-bound polyphenol oxidase from Fuji apple.

    PubMed

    Liu, Fang; Zhao, Jin-Hong; Wen, Xin; Ni, Yuan-Ying

    2015-09-15

    Membrane-bound polyphenol oxidase (mPPO) in Fuji apple (Malus domestica Borkh. cv. Red Fuji) was purified and analyzed with a nanoelectrospray ionization mass spectrometer. The three-dimensional model and binding site of mPPO to 4-methyl catechol were also studied using molecular docking. mPPO was purified 54.41-fold using temperature-induced phase partitioning technique and ion exchange chromatography. mPPO had a molecular weight of 67.3kDa. Even though a significant level of homology was observed between mPPO and the soluble polyphenol oxidase in the copper binding sequence, there was another region, rich in histidine residues, which differed in 13 amino acids. The three-dimensional structure of mPPO consisted of six α-helices, two short β-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224 and Trp228 formed hydrogen bonds with 4-methyl-catechol.

  18. Cloning of SEZ-12 encoding seizure-related and membrane-bound adhesion protein.

    PubMed

    Kajiwara, K; Nagasawa, H; Shimizu-Nishikawa, K; Ookura, T; Kimura, M; Sugaya, E

    1996-05-06

    SEZ-12 is one of the seizure-related cDNAs which was isolated by differential hybridization from primary cultured neurons from the mouse cerebral cortex with or without pentylenetetrazol (PTZ). SEZ-12 expression is transiently down-regulated in the mouse brain by injection of PTZ. To characterize SEZ-12, isolation of full-length cDNA and nucleotide sequence analysis were performed. The deduced amino acid sequence of SEZ-12 revealed that it encodes membrane-bound C-type lectin and has a significant homology to that of human cDNA, DGCR2 and IDD, which were cloned from a balanced translocation breakpoint associated with the DiGeorge syndrome. The isolated cDNA was about 4 kb in length and the message was expressed ubiquitously in various organs with low-abundance. Previously, we also cloned a transmembrane protein which is probably involved in cell-cell interaction by the differential hybridization technique. These findings suggest that transmembrane signaling in neuronal cells may have an important role in PTZ-induced seizure.

  19. A Membrane-bound Hemoglobin from Gills of the Green Shore Crab Carcinus maenas*

    PubMed Central

    Ertas, Beyhan; Kiger, Laurent; Blank, Miriam; Marden, Michael C.; Burmester, Thorsten

    2011-01-01

    Most hemoglobins serve for the transport or storage of O2. Although hemoglobins are widespread in “entomostracan” Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe2+ and an oxygen affinity of P50 = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O2 supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans. PMID:21118803

  20. Structural characterization of membrane-bound human immunodeficiency virus-1 Gag matrix with neutron reflectometry

    PubMed Central

    Eells, Rebecca; Barros, Marilia; Scott, Kerry M.; Karageorgos, Ioannis; Heinrich, Frank; Lösche, Mathias

    2017-01-01

    The structural characterization of peripheral membrane proteins represents a tremendous challenge in structural biology due to their transient interaction with the membrane and the potential multitude of protein conformations during this interaction. Neutron reflectometry is uniquely suited to address this problem because of its ability to structurally characterize biological model systems nondestructively and under biomimetic conditions that retain full protein functionality. Being sensitive to only the membrane-bound fraction of a water-soluble peripheral protein, neutron reflectometry obtains a low-resolution average structure of the protein-membrane complex that is further refined using integrative modeling strategies. Here, the authors review the current technological state of biological neutron reflectometry exemplified by a detailed report on the structure determination of the myristoylated human immunodeficiency virus-1 (HIV-1) Gag matrix associated with phosphoserine-containing model membranes. The authors found that the HIV-1 Gag matrix is able to adopt different configurations at the membrane in a pH-dependent manner and that the myristate group orients the protein in a way that is conducive to PIP2-binding. PMID:28511544

  1. Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦

    PubMed Central

    Bellomio, Augusto; Gil-Cartón, David; Redondo-Morata, Lorena; Sot, Jesús; Scheuring, Simon; Valle, Mikel; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2016-01-01

    Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT. PMID:27445331

  2. Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid," Watasenia scintillans.

    PubMed

    Tsuji, Frederick I

    2002-08-19

    The small Japanese "firefly squid," Watasenia scintillans, emits a bluish luminescence from dermal photogenic organs distributed along the ventral aspects of the head, mantle, funnel, arms and eyes. The brightest light is emitted by a cluster of three tiny organs located at the tip of each of the fourth pair of arms. Studies of extracts of the arm organs show that the light is due to a luciferin-luciferase reaction in which the luciferase is membrane-bound. The other components of the reaction are coelenterazine disulfate (luciferin), ATP, Mg(2+), and molecular oxygen. Based on the results, a reaction scheme is proposed which involves a rapid base/luciferase-catalyzed enolization of the keto group of the C-3 carbon of luciferin, followed by an adenylation of the enol group by ATP. The AMP serves as a recognition moiety for docking the substrate molecule to a luciferase bound to membrane, after which AMP is cleaved and a four-membered dioxetanone intermediate is formed by the addition of molecular oxygen. The intermediate then spontaneously decomposes to yield CO(2) and coelenteramide disulfate (oxyluciferin) in the excited state, which serves as the light emitter in the reaction.

  3. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes.

    PubMed

    Coleman, R A; Rao, P; Fogelsong, R J; Bardes, E S

    1992-04-23

    2-Bromopalmitate and 2-bromopalmitoyl-CoA have been shown to inhibit a variety of enzymes and proteins associated with lipid metabolism. We found that both of the brominated compounds were non-competitive inhibitors of two microsomal activities of triacylglycerol biosynthesis, the mono- and diacylglycerol acyltransferases. With both compounds, the calculated Ki values were lower than the Km value for the palmitoyl-CoA substrate. In addition to inhibiting two other lipid synthetic activities, fatty acid CoA ligase and glycerol-3-P acyltransferase, 2-bromopalmitate and 2-bromopalmitoyl-CoA also inhibited two microsomal enzyme activities that are not related to lipid metabolism, NADPH cytochrome-c reductase and glucose-6-phosphatase. Inhibition of the three acyltransferases and fatty acid CoA ligase could be overcome by the addition of phospholipid vesicles, and 2-bromo[14C]palmitate readily labeled a large number of membrane-bound proteins as well as cytosolic proteins that had been solubilized in SDS. Thus, it appears likely that the inhibitory properties of the brominated compounds strongly depend on the effective concentration of the inhibitor within membranes rather than on any specific affinity for an acyl-chain binding region of the enzyme.

  4. EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation.

    PubMed

    Hoernke, Maria; Mohan, Jagan; Larsson, Elin; Blomberg, Jeanette; Kahra, Dana; Westenhoff, Sebastian; Schwieger, Christian; Lundmark, Richard

    2017-02-21

    The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

  5. Development of a Membrane-Bound Random DNA Sequence Combinatorial Array Recognition Surface (CARS)

    PubMed Central

    Bruno, John G.

    2010-01-01

    A partially overlapping population of random sequence 60mer DNA molecules consisting of many concatamers of varied lengths was spatially separated in one and two dimensions by electrophoresis in polyacrylamide and transferred to nitrocellulose membranes. The spatially separated library serves as a potential sensor interface on which many different molecular recognition events or target analyte-binding patterns may emerge, thereby theoretically representing a “universal sensor” surface. The separated DNA library has been referred to as a DNA combinatorial array recognition surface or “CARS.” After UV baking and various fluorescence staining or fluorescent probe interactions, the one-dimensional (1-D) and 2-D membrane-bound CARS were digitally photographed and subjected to image analysis with National Institutes of Health Image-Java software. Image analysis demonstrated relatively consistent and more similar spatial fluorescence patterns within CARS analyte treatment groups but noteworthy pattern differences before and after analyte addition and between different analyte treatments. Taken together, these data suggest a potential role for CARS as a novel, inexpensive, self-assembling universal molecular recognition surface that could be coupled to sophisticated Bayesian or other pattern recognition algorithms to classify analytes or make specific identifications, much like the senses of smell or taste. PMID:20357981

  6. Expression of membrane-bound burst-promoting activity is mediated by allogeneic effector cells.

    PubMed

    Guha, A; Tuck, D; Sorba, S; Dainiak, N

    1993-09-01

    To investigate whether "self" and "non-self" recognition processes are involved in murine erythropoiesis, the expression of membrane-bound burst-promoting activity (mBPA) was determined for B lymphocytes purified from spleens of CF-1, C57 BL/6J, B6021-7115, and CAF-1J mice using syngeneic and allogeneic bone marrow cultures. Addition of B lymphocyte conditioned medium (LCM), shed membrane-derived vesicles, or intact plasma membranes prepared from syngeneic murine cells stimulated erythroid burst-forming unit (BFU-E) proliferation by two- to three-fold above control levels. BFU-E proliferation was increased by six- to eight-fold, however, when LCM, shed membrane vesicles, or plasma membranes purified from allogenic B lymphocytes were used as sources of growth-stimulatory activity. Bioactivity was immunoprecipitated from detergent extracts of membranes purified from both allogeneic and syngeneic lymphocytes with a monoclonal antibody that specifically recognizes mBPA, suggesting that the factors expressed by these cells share antigenic determinants. The results indicate that allogeneic effector cells are a more potent source of mBPA-like molecules than are syngeneic cells, suggesting that immune mechanisms may be involved in inducing erythroid growth factor expression at the B cell surface.

  7. Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis

    PubMed Central

    Li, Shengben; Le, Brandon; Ma, Xuan; Li, Shaofang; You, Chenjiang; Yu, Yu; Zhang, Bailong; Liu, Lin; Gao, Lei; Shi, Ting; Zhao, Yonghui; Mo, Beixin; Cao, Xiaofeng; Chen, Xuemei

    2016-01-01

    Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER. DOI: http://dx.doi.org/10.7554/eLife.22750.001 PMID:27938667

  8. A membrane-bound NAC transcription factor as an integrator of biotic and abiotic stress signals.

    PubMed

    Seo, Pil Joon; Park, Chung-Mo

    2010-05-01

    Transcription factors are central components of gene regulatory networks that mediate virtually all aspects of growth and developmental processes in biological systems. The activity of transcription factors is regulated at multiple steps, such as gene transcription, posttranscriptional RNA processing, posttranslational modification, protein-protein interactions, and controlled protein turnover. Controlled activation of dormant, membrane-bound transcription factor (MTF) is an intriguing regulatory mechanism that ensures quick transcriptional responses to environmental fluctuations in plants, in which various stress hormones serve as signaling mediators. NTL6 is proteolytically activated upon exposure to cold and induces expression of the Pathogenesis-Related (PR) genes. The membrane-mediated cold signaling in inducing pathogen resistance is considered to be an adaptive strategy that protects plants against infection by hydrophilic pathogens frequently occurring during cold season. We found that NTL6 also mediates abscisic acid (ABA) regulation of abiotic stress responses in Arabidopsis. NTL6 is proteolytically activated by ABA. Transgenic plants overexpressing a nuclear NTL6 form (35S:6ΔC) exhibited a hypersensitive response to ABA and high salinity in seed germination. Taken together, these observations indicate that NTL6 plays an integrative role in plant responses to both biotic and abiotic stress conditions.

  9. Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain.

    PubMed

    Jeohn, G H; Matsuzaki, H; Takahashi, K

    1999-03-01

    A detergent-requiring metalloendopeptidase cleaving a progastrin-C-terminal peptide (progastrin-(88-101)) mainly at the Arg95-Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of approximately 76 kDa as estimated by both SDS/PAGE and Sephacryl S-300 gel filtration. It hydrolyzed progastrin-(88-101) peptide, BAM-12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X-100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal-chelating agents such as EDTA, EGTA, and o-phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 microM EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn2+, CO2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A-(1-13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane-bound metalloendopeptidase.

  10. Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function

    PubMed Central

    Snead, David; Lai, Alex L.; Wragg, Rachel T.; Parisotto, Daniel A.; Ramlall, Trudy F.; Dittman, Jeremy S.; Freed, Jack H.; Eliezer, David

    2017-01-01

    Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo. PMID:28596722

  11. Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus.

    PubMed

    Pinkwart, M; Schneider, K; Schlegel, H G

    1983-06-29

    The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.

  12. Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal β-glucocerebrosidase.

    PubMed

    Abdul-Hammed, Misbaudeen; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2017-03-01

    Glucosylceramide (GlcCer) is the primary storage lipid in the lysosomes of Gaucher patients and a secondary one in Niemann-Pick disease types A, B, and C. The regulatory roles of lipids on the hydrolysis of membrane bound GlcCer by lysosomal β-glucocerebrosidase (GBA1) was probed using a detergent-free liposomal assay. The degradation rarely occurs at uncharged liposomal surfaces in the absence of saposin (Sap) C. However, anionic lipids stimulate GlcCer hydrolysis at low pH by up to 1,000-fold depending on the nature and position of the negative charges in their head groups while cationic lipids inhibit the degradation, thus showing the importance of electrostatic interactions between the polycationic GBA1 and the negatively charged vesicle surfaces at low pH. Ceramide, fatty acids, monoacylglycerol, and diacylglycerol also stimulate GlcCer hydrolysis while SM, sphingosine, and sphinganine play strong inhibitory roles, thereby explaining the secondary storage of GlcCer in Niemann-Pick diseases. Surprisingly, cholesterol stimulates GlcCer degradation in the presence of bis(monoacylglycero)phosphate (BMP). Sap C strongly stimulates GlcCer hydrolysis even in the absence of BMP and the regulatory roles of the intraendolysosomal lipids on its activity is discussed. Our data suggest that these strong modifiers of GlcCer hydrolysis affect the genotype-phenotype correlation in several cases of Gaucher patients independent of the types. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  13. The putative roles of nuclear and membrane-bound progesterone receptors in the female reproductive tract.

    PubMed

    Kowalik, Magdalena K; Rekawiecki, Robert; Kotwica, Jan

    2013-12-01

    Progesterone produced by the corpus luteum (CL) is a key regulator of normal cyclical reproductive functions in the females of mammalian species. The physiological effects of progesterone are mediated by the canonical genomic pathway after binding of progesterone to its specific nuclear progesterone receptor (PGR), which acts as a ligand-activated transcription factor and has two main isoforms, PGRA and PGRB. These PGR isoforms play different roles in the cell; PGRB acts as an activator of progesterone-responsive genes, while PGRA can inhibit the activity of PGRB. The ratio of these isoforms changes during the estrous cycle and pregnancy, and it corresponds to the different levels of progesterone signaling occurring in the reproductive tract. Progesterone exerts its effects on cells also by a non-genomic mechanism by the interaction with the progesterone-binding membrane proteins including the progesterone membrane component (PGRMC) 1 and 2, and the membrane progestin receptors (mPRs). These receptors rapidly activate the appropriate intracellular signal transduction pathways, and subsequently they can initiate specific cell responses or modulate genomic cell responses. The diversity of progesterone receptors and their cellular actions enhances the role of progesterone as a factor regulating the function of the reproductive system and other organs. This paper deals with the possible involvement of nuclear and membrane-bound progesterone receptors in the function of target cells within the female reproductive tract.

  14. Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope

    PubMed Central

    Musse, Abdiwahab A.; Boggs, Joan M.; Harauz, George

    2006-01-01

    The degradation of myelin in the CNS is the hallmark of multiple sclerosis. Reduction in the net positive charge of myelin basic protein (MBP), through deimination, correlates strongly with disease severity and may mediate myelin instability and loss of compaction. Using Cys scanning, spin labeling, EPR spectroscopy, and site-specific proteolysis, we show that in the membrane-bound state the primary immunodominant epitope, V83-T92, of the less cationic recombinant murine MBP C8 mimic (rmC8) forms a more highly surface-exposed and shorter amphipathic α-helix than in the unmodified form, recombinant murine MBP C1 mimic (rmC1), analogous to the most cationic and abundant isomer of MBP in normal myelin. Moreover, cathepsin D digested lipid-associated rmC8 3-fold faster than rmC1, and cleavage at F86–F87 occurred more readily in rmC8 than rmC1. These findings suggest a mechanism for initial loss of myelin stability and the autoimmune pathogenesis of multiple sclerosis. PMID:16537438

  15. Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs

    PubMed Central

    Karl, Alexandra; Berner, Arne; Schmitz, Paul; Koch, Matthias; Nerlich, Michael; Mueller, Michael B.

    2016-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism. PMID:27843458

  16. Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs.

    PubMed

    Pfeifer, Christian G; Karl, Alexandra; Berner, Arne; Zellner, Johannes; Schmitz, Paul; Loibl, Markus; Koch, Matthias; Angele, Peter; Nerlich, Michael; Mueller, Michael B

    2016-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism.

  17. An Application of Kalman Filtering to Torpedo Tracking.

    DTIC Science & Technology

    1980-09-01

    pulse transit times from the tor- pedo to a receiving hydrophone array. These measurements, which are nonlinear functions of the position and depth...3000. X-POSIT[ON (FT) Figure 4. True Trajectory of the Torpedo in the Area of a Single Array 47 LL_ 000 - x - y Note: The R G Y error de- creases on...12. True Trajectory of the Torpedo During a Straight Run in the Area of a Single Array 55 000 -x xxX - y U-. - CPA Inbound Leg 4 Outbound Leg - L- 0

  18. Inhibition of the voltage-dependent chloride channel of Torpedo electric organ by diisopropylfluorophosphate and its reversal by oximes

    SciTech Connect

    Abalis, I.M.; Chiang, P.K.; Wirtz, R.A.; Andre, R.G.

    1986-05-01

    Diisopropylfluorophosphate (DFP), a potent organophosphate inhibitor of cholinesterases, was found to inhibit the specific binding of (/sup 35/S)t-butylbicyclophosphorothionate (TBPS), specific chloride channels ligand, to the electric organ membranes of Torpedo, with a Ki of 21 +/- 3 ..mu..M. The binding sites of (/sup 35/S)TBPS in the Torpedo membranes were found not to be GABA receptors or nicotinic acetylcholine receptors as previously described. Interestingly, a stimulation of the binding of (/sup 35/S)TBPS was observed in the presence of atropine and three oximes, monopyridinium oxime 2-PAM, bispyridinium bis-oxime TMB-4 and H-oxime HI-6. The maximal stimulation was 300-500% of control, after which, the stimulation was reversed at higher concentrations. The three oximes protected by more than 95% the inhibition by 1 mM DFP of the binding of (/sup 35/S)TBPS to the voltage-dependent chloride channel. However, atropine protected only 20% of the inhibited channel. These results, thus, suggest that the protection against the toxic effects of DFP or other anticholinesterase agents by the tested oximes may not be solely a result of the reactivation of cholinesterases but also the protection of the voltage-dependent chloride channel.

  19. Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

    PubMed

    Randall, Linnell B; Dobos, Karen; Papp-Wallace, Krisztina M; Bonomo, Robert A; Schweizer, Herbert P

    2015-12-28

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei

    PubMed Central

    Randall, Linnell B.; Dobos, Karen; Papp-Wallace, Krisztina M.; Bonomo, Robert A.

    2015-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose. PMID:26711764

  1. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management

    PubMed Central

    Croyle, Kaitlin E.; Durrant, Barbara S.; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm–oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  2. Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

    PubMed

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Ahuja, Shivani; Pichumani, Kumar; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2012-06-21

    Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions are -187.9, -166.0, and -161.1 ppm, respectively, with an overall average value of -171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb(5). However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.

  3. Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans.

    PubMed

    Meyer, Maria; Schweiger, Paul; Deppenmeier, Uwe

    2013-04-01

    The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.

  4. Detection of oocyte perivitelline membrane-bound sperm: a tool for avian collection management.

    PubMed

    Croyle, Kaitlin E; Durrant, Barbara S; Jensen, Thomas

    2015-01-01

    The success and sustainability of an avian breeding programme depend on managing productive and unproductive pairs. Given that each breeding season can be of immeasurable importance, it is critical to resolve pair fertility issues quickly. Such problems are traditionally diagnosed through behavioural observations, egg lay history and hatch rates, with a decision to re-pair generally taking one or more breeding seasons. In pairs producing incubated eggs that show little or no signs of embryonic development, determining fertility is difficult. Incorporating a technique to assess sperm presence on the oocyte could, in conjunction with behaviour and other data, facilitate a more timely re-pair decision. Detection of perivitelline membrane-bound (PVM-bound) sperm verifies successful copulation, sperm production and sperm functionality. Alternatively, a lack of detectable sperm, at least in freshly laid eggs, suggests no mating, lack of sperm production/function or sperm-oviduct incompatibility. This study demonstrated PVM-bound sperm detection by Hoechst staining in fresh to 24-day-incubated exotic eggs from 39 species representing 13 orders. However, a rapid and significant time-dependent loss of detectable PVM-bound sperm was observed following incubation of chicken eggs. The PCR detection of sperm in seven species, including two bacterially infected eggs, demonstrated that this method was not as reliable as visual detection using Hoechst staining. The absence of amplicons in visually positive PVMs was presumably due to large PVM size and low sperm count, resulting in DNA concentrations too low for standard PCR detection. In summary, this study demonstrated the feasibility and limitations of using PVM-bound sperm detection as a management tool for exotic avian species. We verified that sperm presence or absence on fluorescence microscopy can aid in the differentiation of fertile from infertile eggs to assist breeding managers in making prompt decisions for pair

  5. Molecular characterization of soluble and membrane-bound trehalases of the whitefly, Bemisia tabaci.

    PubMed

    Wang, Jia; He, Wen-Bo; Su, Yun-Lin; Bing, Xiao-Li; Liu, Shu-Sheng

    2014-04-01

    Trehalases (Tres) have been demonstrated to be the key enzymes that are involved in various trehalose-associated physiological processes in insects. However, little attention has been devoted to the Tres in the whitefly, Bemisia tabaci. In this study, a soluble Tre (BtTre-1) and a membrane-bound Tre (BtTre-2) were cloned in the invasive cryptic species Middle East-Asia Minor 1 (MEAM1) of the whitefly B. tabaci complex. Alignment of deduced amino acids sequences of both BtTres revealed that they share common consensus regions and residues with Tres of other insect species. Levels of BtTres expression in various stages and tissues of the whitefly suggested that BtTre-2 may play a key role in trehalose catabolism during development of the whitefly, especially for oocyte development, while BtTre-1 may prevent trehalose in salivary gland from leaking and entering into plants along with saliva. Potential roles of trehalose catabolism in response to direct and/or plant-mediated indirect effects of Tomato Yellow Leaf Curl China Virus (TYLCCNV) were also detected. Whiteflies feeding on virus-infected tobacco plants showed higher BtTres expressions and accordingly higher BtTres activity but lower trehalose content than those feeding on uninfected plants. The enhanced trehalose catabolism may be beneficial to oocyte development in ovary and attenuate plant defensive responses induced by trehalose in saliva. Viruliferous and nonviruliferous whiteflies feeding on cotton, a nonhost plant for TYLCCNV, differed significantly only in trehalose content. The higher trehalose content in viruliferous whiteflies may be conducive to resisting the stress inflicted by TYLCCNV. © 2014 Wiley Periodicals, Inc.

  6. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons.

    PubMed

    Gao, Jie; Naughton, Sean X; Wulff, Heike; Singh, Vikrant; Beck, Wayne D; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M; Terry, Alvin V

    2016-03-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons

    PubMed Central

    Gao, Jie; Naughton, Sean X.; Wulff, Heike; Singh, Vikrant; Beck, Wayne D.; Magrane, Jordi; Thomas, Bobby; Kaidery, Navneet Ammal; Hernandez, Caterina M.

    2016-01-01

    The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs. PMID:26718240

  8. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Sumikawa, K; Houghton, M; Smith, J C; Bell, L; Richards, B M; Barnard, E A

    1982-01-01

    A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle. Images PMID:6183641

  9. Abnormal gene expression of proinflammatory cytokines and their membrane-bound receptors in the lymphocytes of depressed patients.

    PubMed

    Rizavi, Hooriyah S; Ren, Xinguo; Zhang, Hui; Bhaumik, Runa; Pandey, Ghanshyam N

    2016-06-30

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of depressed patients. In this study, we examined the role of cytokines and their membrane-bound receptors in major depressive disorder (MDD). We determined the protein and mRNA expression of proinflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and mRNA expression of their membrane-bound receptors in the lymphocytes from 31 hospitalized MDD patients and 30 non-hospitalized normal control (NC) subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mean mRNA levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, their receptors, TNFR1, TNFR2, IL-1R1 and the antagonist IL-1RA were significantly increased in the lymphocytes of MDD patients compared with NC. No significant differences in the lymphocyte mRNA levels of IL-1R2, IL-6R, and Gp130 were observed between MDD patients and NC. These studies suggest abnormal gene expression of these cytokines and their membrane-bound receptors in the lymphocytes of MDD patients, and that their mRNA expression levels in the lymphocytes could be a useful biomarker for depression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Intracellular localization of membrane-bound ATPases in the compartmentalized anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’

    PubMed Central

    van Niftrik, Laura; van Helden, Mary; Kirchen, Silke; van Donselaar, Elly G; Harhangi, Harry R; Webb, Richard I; Fuerst, John A; Op den Camp, Huub J M; Jetten, Mike S M; Strous, Marc

    2010-01-01

    Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism. PMID:20545867

  11. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

  12. Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport of Methanosarcina mazei Gö1

    PubMed Central

    Abken, Hans-Jörg; Tietze, Mario; Brodersen, Jens; Bäumer, Sebastian; Beifuss, Uwe; Deppenmeier, Uwe

    1998-01-01

    A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F420H2 dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2 was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The Km values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1. PMID:9555882

  13. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2010-01-01

    Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

  14. Presence of membrane-bound proteinases that preferentially degrade oxidatively damaged erythrocyte membrane proteins as secondary antioxidant defense.

    PubMed

    Beppu, M; Inoue, M; Ishikawa, T; Kikugawa, K

    1994-11-23

    Human erythrocytes were oxidized with xanthine/xanthine oxidase/ferric ion or ADP/ferric ion at 37 degrees C for several hours. Band 3 protein and spectrin of the oxidized cells were found to be significantly modified as analyzed by radiolabeling with tritiated borohydride. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the xanthine/xanthine oxidase/ferric iron-oxidized cells and subsequent immunoblotting with anti band 3 protein showed that band 3 protein was fragmented into smaller molecular-weight fragments. When the cell membrane obtained from the oxidized cells were incubated at pH 7.4 and 37 degrees C for several hours in the presence of alpha-tocopherol, extensive degradation of band 3 protein and spectrin was observed. Band 3 protein was found to be most susceptible to the degradation. Degradation of band 3 protein was also observed after similar incubation of the membrane from the ADP/ferric ion-oxidized cells. Membrane-bound serine- and metalloproteinases were responsible for the degradation of band 3 protein, because the degradation was remarkably inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, and partially by ethylenediaminetetraacetic acid. Hence, the membrane proteins became susceptible to membrane-bound proteinases by oxidative stress. This observation suggests that these membrane-bound proteinases exist to remove oxidatively damaged proteins from the cell membrane.

  15. Transient Developmental Purkinje Cell Axonal Torpedoes in Healthy and Ataxic Mouse Cerebellum

    PubMed Central

    Ljungberg, Lovisa; Lang-Ouellette, Daneck; Yang, Angela; Jayabal, Sriram; Quilez, Sabrina; Watt, Alanna J.

    2016-01-01

    Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wild-type and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11). This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occurred largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6), and found elevated disease-related torpedo number at 2

  16. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  17. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  18. EXTERIOR VIEW, LOOKING WEST, WITH SWITCHING ENGINE TRANSPORTING TORPEDO LADLE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    EXTERIOR VIEW, LOOKING WEST, WITH SWITCHING ENGINE TRANSPORTING TORPEDO LADLE FILLED WITH MOLTEN IRON TO THE Q-BOP FURNACES. VERTICAL CONVEYOR TRANSPORTS ALL PROCESS MATERIAL INCLUDING COKE AND PELLETS TO FURNACE. - U.S. Steel, Fairfield Works, Blast Furnace No. 8, North of Valley Road, West of Ensley-Pleasant Grove Road, Fairfield, Jefferson County, AL

  19. Structural and Dynamical Insights into the Membrane-Bound α-Synuclein

    PubMed Central

    Mukhopadhyay, Samrat

    2013-01-01

    Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson’s disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å) layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could potentially be

  20. Structure and promoter analysis of the mouse membrane-bound transferrin-like protein (MTf) gene.

    PubMed

    Nakamasu, K; Kawamoto, T; Yoshida, E; Noshiro, M; Matsuda, Y; Kato, Y

    2001-03-01

    Recently, we purified membrane-bound transferrin-like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem. 256, 503--509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning approximately 26 kb and consisting of 16 exons. The transcription-initiation site was located 157 bp upstream from the translation-start codon, and a TATA box was not found in the 5' flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK-MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5' flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5' deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtained with primary chondrocytes suggest that the regions between -693 and -444 and between -1635 and -1213 contain positive and negative cis-acting elements, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in

  1. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

    PubMed Central

    Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

    1980-01-01

    A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629

  2. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    PubMed Central

    Manetta, Joseph; Bina, Holly; Ryan, Paul; Fox, Niles; Witcher, Derrick R; Kikly, Kristine

    2014-01-01

    B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. PMID:25258549

  3. Membrane-Bound Structure and Topology of a Human Alpha Defensin Indicates A Dimer Pore Mechanism for Membrane Disruption

    PubMed Central

    Zhang, Yuan; Lu, Wuyuan; Hong, Mei

    2010-01-01

    Defensins are cationic and disulfide-bonded host defense proteins of many animals that target microbial cell membranes. Elucidating the three-dimensional structure, dynamics and topology of these proteins in phospholipid bilayers is important for understanding their mechanisms of action. Using solid-state NMR spectroscopy, we have now determined the conformation, dynamics, oligomeric state and topology of a human α-defensin, HNP-1, in DMPC/DMPG bilayers. 2D correlation spectra show that membrane-bound HNP-1 exhibits a similar conformation to the water-soluble state, except for the turn connecting the β2 and β3 strands, whose sidechains exhibit immobilization and conformational perturbation upon membrane binding. At high protein/lipid ratios, rapid 1H spin diffusion from the lipid chains to the protein was observed, indicating that HNP-1 was well inserted into the hydrocarbon core of the bilayer. Arg Cζ-lipid 31P distances indicate that only one of the four Arg residues forms tight hydrogen-bonded guanidinium-phosphate complexes. The protein is predominantly dimerized at high protein/lipid molar ratios, as shown by 19F spin diffusion experiments. The presence of a small fraction of monomers and the shallower insertion at lower protein concentrations suggest that HNP-1 adopts concentration-dependent oligomerization and membrane-bound structure. These data strongly support a “dimer pore” topology of HNP-1 in which the polar top of the dimer lines an aqueous pore while the hydrophobic bottom faces the lipid chains. In this structure R25 lies closest to the membrane surface among the four Arg residues. The pore does not have large lipid disorder, in contrast to the toroidal pores formed by protegrin-1, a two-stranded β-hairpin antimicrobial peptide. These results provide the first glimpse into the membrane-bound structure and mechanism of action of human α-defensins. PMID:20961099

  4. Mass Spectrometric Detection and Characterization of Atypical Membrane-Bound Zinc-Sensitive Phosphatases Modulating GABAA Receptors

    PubMed Central

    SidAhmed-Mezi, Mounia; Kurcewicz, Irène; Rose, Christiane; Louvel, Jacques; Sokoloff, Pierre; Pumain, René; Laschet, Jacques J.

    2014-01-01

    Background GABAA receptor (GABAAR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I2 of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABAAR are deficient. Methodology/Principal Findings The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABAAR. We have developed a new analytical tool for in vitro detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABAAR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABAAR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABAAR. Conclusion/Significance Our results provide new insights on the regulation of GABAAR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed. PMID:24967814

  5. Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy*

    PubMed Central

    Yap, Thai Leong; Jiang, Zhiping; Heinrich, Frank; Gruschus, James M.; Pfefferkorn, Candace M.; Barros, Marilia; Curtis, Joseph E.; Sidransky, Ellen; Lee, Jennifer C.

    2015-01-01

    Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact. PMID:25429104

  6. pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

    PubMed Central

    Gray, C.; Tamm, L. K.

    1998-01-01

    Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined

  7. Isolation and characterization of secretory granules storing a vasoactive intestinal polypeptide-like peptide in Torpedo cholinergic electromotor neurones.

    PubMed

    Agoston, D V; Dowe, G H; Whittaker, V P

    1989-06-01

    Previous immunocytochemical work showed that the cholinergic electromotor neurones of Torpedo marmorata contain a vasoactive intestinal polypeptide-like immunoreactivity (VIPLI) that is conveyed to the terminals by axonal transport from the cell bodies where it is presumably synthesized. In extension of this work, we have now succeeded in isolating the VIPLI storage granules from both the terminals and the axons of these neurones and characterizing them morphologically and biochemically. They were readily separated from synaptic vesicles but contained several components in common that had previously been regarded as specific for synaptic vesicles. Among these were a heparan sulphate type of proteoglycan, synaptophysin, and a Mg2+-dependent ATPase. The VIPLI concentration in lobe tissue and the amount of tissue available were both insufficient to permit the isolation of granules from the electromotor cell bodies by the same technique but it was possible to establish the presence of such granules by particle-exclusion chromatography, using the stable markers mentioned above. In contrast to the VIPLI-containing granules, axonal synaptic vesicles differed from their terminal counterparts in having a very low acetylcholine content relative to stable vesicle markers: they presumably fill up on reaching the terminal where they are exposed to higher concentrations of cytoplasmic acetylcholine.

  8. Acetylcholine Promotes Binding of α-Conotoxin MII for α3β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Sambasivarao, Somisetti V.; Roberts, Jessica; Bharadwaj, Vivek S.; Slingsby, Jason G.; Rohleder, Conrad; Mallory, Chris; Groome, James R.

    2014-01-01

    α-Conotoxin MII (α-CTxMII) is a 16 amino acid peptide with the sequence GCCSNPVCHLEHSNLC containing disulfide bonds between Cys2-Cys8 and Cys3-Cys16. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER using crystal structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3 and β2 subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with commensurate binding energies to previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, which was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. PMID:24420650

  9. Acetylcholine promotes binding of α-conotoxin MII at α3 β2 nicotinic acetylcholine receptors.

    PubMed

    Sambasivarao, Somisetti V; Roberts, Jessica; Bharadwaj, Vivek S; Slingsby, Jason G; Rohleder, Conrad; Mallory, Chris; Groome, James R; McDougal, Owen M; Maupin, C Mark

    2014-02-10

    α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and β2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

    PubMed Central

    LaPolla, R J; Mayne, K M; Davidson, N

    1984-01-01

    A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

  11. Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice.

    PubMed

    Pitari, G; Malergue, F; Martin, F; Philippe, J M; Massucci, M T; Chabret, C; Maras, B; Duprè, S; Naquet, P; Galland, F

    2000-10-20

    Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

  12. Size exclusion chromatography-multiangle laser light scattering analysis of hyaluronan size distributions made by membrane-bound hyaluronan synthase.

    PubMed

    Baggenstoss, Bruce A; Weigel, Paul H

    2006-05-15

    Size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analyses of Escherichia coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10-100 MDa) that coeluted with hyaluronan (HA) and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65-75 degrees C eliminated this artifact and increased the yield of recovered HA due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed uridine 5'-diphosphate (UDP) produced during the reaction, improved the linearity of HA synthesis-even at high substrate use. Surprisingly, the addition of EDTA, to chelate Mg(2+) ions, did not completely stop the HAS reaction at 30 degrees C or at 4 degrees C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4 to 6 MDa within 2-4 h.

  13. Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity

    PubMed Central

    Fusco, Giuliana; De Simone, Alfonso; Arosio, Paolo; Vendruscolo, Michele; Veglia, Gianluigi; Dobson, Christopher M.

    2016-01-01

    A detailed characterisation of the molecular determinants of membrane binding by α-synuclein (αS), a 140-residue protein whose aggregation is associated with Parkinson’s disease, is of fundamental significance to clarify the manner in which the balance between functional and dysfunctional processes are regulated for this protein. Despite its biological relevance, the structural nature of the membrane-bound state αS remains elusive, in part because of the intrinsically dynamic nature of the protein and also because of the difficulties in studying this state in a physiologically relevant environment. In the present study we have used solid-state NMR and restrained MD simulations to refine structure and topology of the N-terminal region of αS bound to the surface of synaptic-like membranes. This region has fundamental importance in the binding mechanism of αS as it acts as to anchor the protein to lipid bilayers. The results enabled the identification of the key elements for the biological properties of αS in its membrane-bound state. PMID:27273030

  14. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis.

  15. Mg2+ is an essential activator of hydrolytic activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum.

    PubMed Central

    Sosa, A; Ordaz, H; Romero, I; Celis, H

    1992-01-01

    The substrate for the hydrolytic activity of membrane-bound pyrophosphatase is the PP(i)-Mg2+ complex. The enzyme has no activity when the free Mg2+ concentration is lower than 10 microM (at 0.5 mM-PP(i)-Mg2+), and therefore free Mg2+ is an essential activator of the hydrolytic activity. The Km for the substrate changes in response to variation in free Mg2+ concentration, from 10.25 to 0.6 mM when free Mg2+ is increased from 0.03 to 1.0 mM respectively. The Km for Mg2+ depends on the substrate concentration: the Km decreases from 0.52 to 0.14 mM from 0.25 to 0.75 mM-PP(i)-Mg2+ respectively. The extrapolated Km for Mg2+ in the absence of the substrate is 0.73 mM. Imidodiphosphate-Mg2+ and free Ca2+ were used as competitive inhibitors of substrate and activator respectively. The equilibrium binding kinetics suggest an ordered mechanism for the activator and the substrate: Mg2+ ions bind the enzyme before PP(i)-Mg2+ in the formation of the catalytic complex, membrane-bound pyrophosphatase-(Mg2+)-(PP(i)-Mg2+). PMID:1315519

  16. Are hormones from the neuropeptide Y family recognized by their receptors from the membrane-bound state?

    PubMed

    Bader, Reto; Zerbe, Oliver

    2005-09-01

    Hormones and many other neurotransmitters, growth factors, odorant molecules, and light all present stimuli for a class of membrane-anchored receptors called G protein-coupled receptors (GPCRs). The GPCRs are the largest family of cell-surface receptors involved in signal transduction. About 1% of all known genes of Drosophila and more than 5% of the genes of Caenorhabditis elegans encode GPCRs. In addition, more than 50% of current therapeutic agents on the market target these receptors. When the enormous biological and pharmaceutical importance of these receptors is considered, it is surprising how little is known about the mechanism with which these receptors recognize their natural ligands. In this review we present a structural approach, utilizing techniques of high-resolution NMR spectroscopy, to address the question of whether peptides from the neuropeptide Y family of neurohormones are recognized directly from solution or from the membrane-bound state. In our studies we discovered that the structures of the membrane-bound species are better correlated to the pharmacological properties of these peptides than the solution structures are. These findings are supported by the observation that many biophysical properties of these peptides seem to be optimized for membrane binding. We finally present a scenario of possible events during receptor recognition.

  17. Protective Effect of Prosopis cineraria Against N-Nitrosodiethylamine Induced Liver Tumor by Modulating Membrane Bound Enzymes and Glycoproteins

    PubMed Central

    Pakkir Maideen, Naina Mohamed; Velayutham, Ravichandiran; Manavalan, Gobinath

    2012-01-01

    Purpose: The objective of the present study was to evaluate the protective effect of methanol extract of Prosopis cineraria (MPC) against N-nitrosodiethylamine (DEN, 200mg/kg) induced Phenobarbital promoted experimental liver tumors in male Wistar rats. Methods: The rats were divided into four groups, each group consisting of six animals. Group 1 served as control animals. Liver tumor was induced in group 2, 3, and 4 and Group 3 animals received MPC 200mg/kg and Group 4 animals received MPC 400mg/kg. Results: Administration of DEN has brought down the levels of membrane bound enzymes like Na+/ K+ ATPase, Mg2+ ATPase and Ca2+ATPase which were later found to be increased by the administration of Prosopis cineraria (200 and 400mg/kg) in dose dependent manner. The MPC extract also suppressed the levels of glycoproteins like Hexose, Hexosamine and Sialic acid when compared to liver tumor bearing animals. Conclusion: Our study suggests that MPC may extend its protective role by modulating the levels of membrane bound enzymes and suppressing glycoprotein levels. PMID:24312790

  18. Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate.

    PubMed

    Manzo, Anthony J; Goushcha, Alexander O; Barabash, Yuri M; Kharkyanen, Valery N; Scott, Gary W

    2009-07-01

    Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I(exp), obeys a simple exponential law with the rate constant alphaI(exp) + k(rec), in which alpha is a parameter relating the light intensity, measured in mW/cm(2), to a corresponding theoretical rate in units of reciprocal seconds, and k(rec) is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the alpha parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer-Lambert-Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

  19. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-11-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.

  20. Activity of soluble aminopeptidase A and dipeptidyl peptidase IV and membrane-bound aminopeptidase B and pyroglutamyl peptidase I in adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis.

    PubMed

    Larrinaga, Gorka; Perez, Itxaro; Sanz, Begoña; Irazusta, Amaya; Zarrazquin, Idoia; Sanchez, Clara Elena; Sanchez, Carmen Elena; Rey, Ana Sanchez Del; Zabala, Aitor; Santaolalla, Francisco

    2011-11-01

    To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis. A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically. The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p=0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, p<0.05, while membrane-bound aminopeptidase B activity was higher in tissues of females with tonsillar hyperplasia, p<0.001. In the case of chronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p=0.005. Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  2. 33 CFR 334.1190 - Hood Canal and Dabob Bay, Wash.; naval non-explosive torpedo testing area.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ....; naval non-explosive torpedo testing area. 334.1190 Section 334.1190 Navigation and Navigable Waters... REGULATIONS § 334.1190 Hood Canal and Dabob Bay, Wash.; naval non-explosive torpedo testing area. (a) Hood... are about to enter the area while a torpedo is in the water will be contacted by a Navy patrol boat...

  3. Effect of Butanedioic Acid Mono (2,2-Dimethylhydrazide) on the Activity of Membrane-Bound Succinate Dehydrogenase

    PubMed Central

    See, Raymond M.; Foy, Chester L.

    1982-01-01

    Mitochondria isolated from hypocotyls of five-day-old bean (Phaseolus vulgaris L. `Black Valentine') seedlings rapidly oxidized succinate, malate, and NADH. Oxidation rates, respiratory control, and ADP:O ratios obtained with saturating concentrations of all three substrates indicated that the mitochondria were tightly coupled. The mitochondrial preparation was then employed to investigate the respiration-inhibiting effects of butanedioic acid mono (2,2-dimethyl-hydrazide) (daminozide) a plant growth retardant having structural similarity to an endogenous respiratory substrate (succinate). Daminozide markedly inhibited the activity of membrane-bound succinate dehydrogenase. Inhibition was of the competitive type (apparent Ki, 20.2 millimolar) with respect to succinate. Although not excluding other hypotheses, the results support an active role for daminozide in the suppression of respiration as an important metabolic site of its action as a plant growth regulator. PMID:16662493

  4. Penconazole alters redox status, cholinergic function, and membrane-bound ATPases in the cerebrum and cerebellum of adult rats.

    PubMed

    Chaâbane, M; Ghorbel, I; Elwej, A; Mnif, H; Boudawara, T; Chaâbouni, S Ellouze; Zeghal, N; Soudani, N

    2016-10-12

    Pesticides exposure causes usually harmful effects to the environment and human health. The present study aimed to investigate the potential toxic effects of penconazole, a triazole fungicide, on the cerebrum and cerebellum of adult rats. Penconazole was administered intraperitoneally to male Wistar rats at a dose of 67 mg kg(-1) body weight every 2 days during 9 days. Results showed that penconazole induced oxidative stress in rat cerebrum and cerebellum tissues. In fact, we have found a significant increase in malondialdehyde, hydrogen peroxide, and advanced oxidation protein product levels, as well as an alteration of the antioxidant status, enzymatic (superoxide dismutase and catalase) and nonenzymatic (glutathione), the cholinergic function, and membrane-bound ATPases (Na(+)/K(+)-ATPase and Mg(2+)-ATPase). Penconazole also provoked histological alterations marked by pyknotic and vacuolated neurons in the cerebrum and apoptosis and edema in the cerebellum Purkinje cells' layer. Therefore, the use of this neurotoxicant fungicide must be regularly monitored in the environment.

  5. Solubilization, purification, and properties of membrane-bound D-glucono-delta-lactone hydrolase from Gluconobacter oxydans.

    PubMed

    Shinagawa, Emiko; Ano, Yoshitaka; Yakushi, Toshiharu; Adachi, Osao; Matsushita, Kazunobu

    2009-01-01

    Membrane-bound glucono-delta-lactonase (MGL) was purified to homogeneity from the membrane fraction of Gluconobacter oxydans IFO 3244. After solubilization with 1 M CaCl2, MGL was purified in the presence of Ca2+ and detergent. A single band corresponding to 60 kDa appeared in SDS-PAGE. The molecular weight of MGL was judged to be 120 k. Differently from cytoplasmic lactonases, MGL showed optimum pH in an acidic range of 5-5.5. It was highly sensitive to metal-chelating agents such as EDTA, and the lost MGL activity was restored to the original level by the addition of divalent cations such as Ca2+ or Mg2+. The purified MGL was strictly dependent on Ca2+ and underwent rapid denaturing precipitation on Ca2+ depletion even in the presence of detergent. This communication can be the first one dealing with the solubilization, purification and properties of MGL.

  6. Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

    PubMed

    Madduri, Krishna M; Schafer, Barry W; Hasler, James M; Lin, Gaofeng; Foster, Mendy L; Embrey, Shawna K; Sastry-Dent, Lakshmi; Song, Ping; Larrinua, Ignacio M; Gachotte, Daniel J; Herman, Rod A

    2012-10-01

    A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.

  7. Photochemical energy conversion by membrane-bound photoredox systems. Progress report, July 1, 1989--March 1, 1992

    SciTech Connect

    Tollin, G.

    1992-03-01

    Most of our effort during the past grant period has been directed towards investigating electron transfer processes involving redox proteins at lipid bilayer/aqueous interfaces. This theme, as was noted in our previous three year renewal proposal, is consistent with our goal of developing biomimetic solar energy conversion systems which utilize the unique properties of biological electron transfer molecules. Thus, small redox proteins such as cytochrome c, plastocyanin and ferredoxin function is biological photosynthesis as mediators of electron flow between the photochemical systems localized in the membrane, and more complex soluble or membrane-bound redox proteins which are designed to carry out specific biological tasks such as transbilayer proton gradient formation, dinitrogen fixation, ATP synthesis, dihydrogen synthesis, generation of strong reductants, etc. In these studies, we have utilized two principal experimental techniques, laser flash photolysis and cyclic voltammetry, both of which permit direct measurements of electron transfer processes.

  8. The torpedo (DER) receptor tyrosine kinase is required at multiple times during Drosophila embryogenesis.

    PubMed

    Clifford, R; Schüpbach, T

    1992-07-01

    The torpedo (DER) gene of Drosophila, which encodes a receptor tyrosine kinase of the EGF receptor subfamily, is essential for oogenesis, embryogenesis and imaginal disc development. To gain insight into the nature of the signals transduced by the torpedo product, we have characterized the gene's loss-of-function phenotype in the embryo. Through the induction of germline clones, we provide a genetic demonstration that maternal torpedo product does not contribute to zygotic development. Thus, the embryonic lethal phenotypes examined accurately reflect the consequences of eliminating all gene activity from the zygote. Temperature-shift experiments with the conditional allele topIF26 show that torpedo is required at two distinct times during embryonic development: the gene is first needed for germband retraction and for the production of anterior, posterior and ventral cuticle, then later for the secretion of ventral denticles. Since denticle formation can be severely disrupted in topIF26 animals without affecting cuticle production, the early and late requirements for torpedo appear to be functionally unrelated. torpedo, therefore, is required at multiple times in the development of the ventral epidermis, and may transduce qualitatively different signals. Since the early requirement for torpedo correlates with the first visible defect in embryonic development, increased cell death in the amnioserosa, cephalic ectoderm and ventral epidermis, the abnormalities in cuticle production and germband shortening seen in the mutant may be secondary consequences of a primary defect in cell viability. Given that the onset of cell death in torpedo embryos is not preceded by any obvious defects in mitogenesis, the establishment of cell identities or the maintenance of gene expression, it is possible that torpedo transduces a signal necessary for cell survival per se during early embryogenesis. During late embryogenesis, torpedo may mediate the reception of a second signal which

  9. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    PubMed Central

    Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

  10. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions1

    PubMed Central

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-01-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression. PMID:26152359

  11. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    PubMed

    Lundin, Anna; Dijkman, Ronald; Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-05-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  12. Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone1[OPEN

    PubMed Central

    Fraga, Patricia

    2017-01-01

    In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3. In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1. We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer. PMID:27956490

  13. Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

    PubMed Central

    Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

    2010-01-01

    Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

  14. Summary of Investigations of Mark 25 Aerial-Torpedo Turbine

    NASA Technical Reports Server (NTRS)

    Schum, Harold J.; Whitney, Warren J.; Buckner, Howard A., Jr.

    1950-01-01

    The power plant from a Mark 25 aerial torpedo was investigated both as a two-stage turbine and as a single-stage modified turbine to determine the effect on overall performance of nozzle size and shape, first-stage rotor-blade configuration, and axial nozzle-rotor running clearance. Performance was evaluated in terms of brake, rotor, and blade efficiencies. All the performance data were obtained for inlet total to outlet static pressure ratios of 8, 15 (design), and 20 with inlet conditions maintained constant at 95 pounds per square inch gage and 1000 F for rotor speeds from approximately 6000 to 18,000 rpm.

  15. The α-bungarotoxin binding site on the nicotinic acetylcholine receptor: Analysis using a phage–epitope library

    PubMed Central

    Balass, Moshe; Katchalski-Katzir, Ephraim; Fuchs, Sara

    1997-01-01

    The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. α-Neurotoxins, in particular α-bungarotoxin (α-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with α-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to α-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the α-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind α-BTX. The library-derived peptide and the corresponding peptides containing residues 187–199 of the Torpedo AcChoR α-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human α7; ERFYECCKEPYPD) that binds α-BTX, inhibit the binding of α-BTX to the intact Torpedo AcChoR with IC50 values of 10−6 M. A synthetic peptide from a neuronal AcChoR that does not bind α-BTX (e.g., human α2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the α-BTX-binding protein (α7)—i.e., Lys in α2 and Tyr in α7—does not inhibit the binding of α-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to α-BTX. PMID:9177167

  16. Axonal transport in the electromotor nerves of Torpedo marmorata.

    PubMed

    Davies, L P; Whittaker, V P; Zimmermann, H

    1977-12-19

    Studies on the axonal transport of cholinergic cell components were made on the electromotor nerves of Torpedo marmorata. Choline acetyltransferase was rapidly accumulated at ligatures on Torpedo nerves, both in vivo and in segments incubated in vitro. In vivo accumulation was maximal approximately one month after nerve interruption. Orthograde transport (both in vitro and in vivo) is calculated to have a velocity of 50--140 mm/day, if, as double-ligature experiments suggest, only about 15% of the axoplasmic enzyme is mobile. A small retrograde accumulation of the transferase was demonstrated. Lactate dehydrogenase did not accumulate but a slight reduction of its activity at ligatures was observed. In contrast to mammalian cholinergic nerves, no accumulation of esterase was observed. ACh accumulation proximal to a cut was apparent and may result in part from local synthesis in the presence of elevated levels of its synthesizing enzyme. Measurements have been made on the activity of choline acetyltransferase in the brain and all parts of the "electric system". In view of these results it is difficult to see how the measured rate of axonal translocation is sufficient to supply the levels of the enzyme found within the electric organ. Within the electromotor cells, choline acetyltransferase is highly concentrated in the axon terminals.

  17. [Redescription of the species of Calyptrobothrium Monticelli, 1893 (Tetraphyllidea: Phyllobothriidae) parasites of Torpedo marmorata in Torpedo nobiliana (Elasmobranchii: Torpedinidae)].

    PubMed

    Tazerouti, Fadila; Euzet, Louis; Kechemir-Issad, Nadia

    2007-07-01

    A study of the cestodes from the intestinal parasites of torpedinid elasmobranchs off the coast of Algeria, revealed the presence, in Torpedo marmorata Risso, of a species of Calyptrobothrium Monticelli, 1893, which we believe is identical to C. riggii Monticelli, 1893. This species, which has not been re-examined since its original description, is redescribed. We consider that the cestode from T. marmorata, described by Zschokke (1888) under the name of Tetrabothrium longicolle Molin, 1858, is conspecific with C.riggii. In Torpedo nobiliana Bonaparte, from the Mediterranean, we found C. occidentale Linton, 1900 and C. minus Linton, 1907. These two species are redescribed based on the type-material from the North American Atlantic coast, as well as from specimens collected off Sète and Algeria. We place several specimens, previously identified as C. riggii, as conspecific with C. minus. An amended diagnosis for Calyptrobothrium is proposed. We discuss the attribution of Bilocularia hyperapolytica Obersteiner, 1914 from Centrophorus granulosus (Centrophoridae) to Calyptrobothrium. C. chalarosomum Alexander, 1963 from Cephaloscyllium isabellum (Scylliorhinidae) off New-Zealand is excluded from the genus and placed close to Crossobothrium Linton, 1889.

  18. The pore domain of the nicotinic acetylcholine receptor: molecular modeling, pore dimensions, and electrostatics.

    PubMed Central

    Sankararamakrishnan, R; Adcock, C; Sansom, M S

    1996-01-01

    The pore domain of the nicotinic acetylcholine receptor has been modeled as a bundle of five kinked M2 helices. Models were generated via molecular dynamics simulations incorporating restraints derived from 9-A resolution cryoelectron microscopy data (Unwin, 1993; 1995), and from mutagenesis data that identify channel-lining side chains. Thus, these models conform to current experimental data but will require revision as higher resolution data become available. Models of the open and closed states of a homopentameric alpha 7 pore are compared. The minimum radius of the closed-state model is less than 2 A; the minimum radius of the open-state models is approximately 6 A. It is suggested that the presence of "bound" water molecules within the pore may reduce the effective minimum radii below these values by up to approximately 3 A. Poisson-Boltzmann calculations are used to obtain a first approximation to the potential energy of a monovalent cation as it moves along the pore axis. The differences in electrostatic potential energy profiles between the open-state models of alpha 7 and of a mutant of alpha 7 are consistent with the experimentally observed change in ion selectivity from cationic to anionic. Models of the open state of the heteropentameric Torpedo nicotinic acetylcholine receptor pore domain are also described. Relatively small differences in pore radius and electrostatic potential energy profiles are seen when the Torpedo and alpha 7 models are compared. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 PMID:8889144

  19. Membrane-bound IL-22 after de novo production in tuberculosis and anti-M.tuberculosis effector function of IL-22+CD4+ T cells

    PubMed Central

    Zeng, Gucheng; Chen, Crystal Y.; Huang, Dan; Yao, Shuyu; Wang, Richard C.; Chen, Zheng W.

    2013-01-01

    The role of IL-22-producing CD4+ T cells in intracellular pathogen infections is poorly characterized. IL-22-producing CD4+ T cells may also express other effector molecules, and therefore synergize or contribute to anti-microbial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22+ T cells cannot be purified for evaluation due to secretion nature of cytokines. Here, we surprisingly found that upon activation, CD4+ T cells in M. tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22+ CD4+ T effector cells appeared to mature in vivo and sustain membrane distribution in highly-inflammatory environments during active M. tuberculosis infection. NSOM/QD-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ~100–200 nm nanoclusters or ~300–600 nm nanodomains for potential interaction with IL-22 receptor. Importantly, purified membrane-bound IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22-producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 half-lives and to exert efficient cell-cell interaction for anti-M. tuberculosis effector function. PMID:21632708

  20. Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature.

    PubMed

    Liu, Wei; Li, Wei; He, Qiuling; Daud, Muhammad Khan; Chen, Jinhong; Zhu, Shuijin

    2015-01-01

    To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.

  1. Recent Structural and Mechanistic Insights into Endplate Acetylcholine Receptors

    PubMed Central

    Sine, Steven M.; Gao, Fan; Lee, Won Yong; Mukhtasimova, Nuriya; Wang, Hai-Long; Engel, Andrew G.

    2012-01-01

    Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms. PMID:18567853

  2. Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase activities.

    PubMed

    Fan, Ming Z; Adeola, Olayiwola; Asem, Elikplimi K; King, Dale

    2002-07-01

    Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.

  3. Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

    PubMed Central

    Strandberg, Erik; Verdurmen, Wouter P. R.; Bürck, Jochen; Ehni, Sebastian; Mykhailiuk, Pavel K.; Afonin, Sergii; Gerthsen, Dagmar; Komarov, Igor V.; Brock, Roland; Ulrich, Anne S.

    2014-01-01

    Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for 19F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N

  4. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

    PubMed

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-06-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Differential Expression of Growth Factor Receptors and Membrane-Bound Tumor Markers for Imaging in Male and Female Breast Cancer

    PubMed Central

    Vermeulen, Jeroen F.; Kornegoor, Robert; van der Wall, Elsken; van der Groep, Petra; van Diest, Paul J.

    2013-01-01

    Introduction Male breast cancer accounts for 0.5–1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. Methods Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. Results Growth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. Conclusions Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for

  6. Differential expression of growth factor receptors and membrane-bound tumor markers for imaging in male and female breast cancer.

    PubMed

    Vermeulen, Jeroen F; Kornegoor, Robert; van der Wall, Elsken; van der Groep, Petra; van Diest, Paul J

    2013-01-01

    Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer. Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers. Growth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers. Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for male breast cancer.

  7. Individual synaptic vesicles from the electroplaque of Torpedo californica, a classic cholinergic synapse, also contain transporters for glutamate and ATP

    PubMed Central

    Li, Huinan; Harlow, Mark L.

    2014-01-01

    Abstract The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single‐vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters – and potentially neurotransmitters – in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses. PMID:24744885

  8. An electric generator using living Torpedo electric organs controlled by fluid pressure-based alternative nervous systems.

    PubMed

    Tanaka, Yo; Funano, Shun-Ichi; Nishizawa, Yohei; Kamamichi, Norihiro; Nishinaka, Masahiro; Kitamori, Takehiko

    2016-05-31

    Direct electric power generation using biological functions have become a research focus due to their low cost and cleanliness. Unlike major approaches using glucose fuels or microbial fuel cells (MFCs), we present a generation method with intrinsically high energy conversion efficiency and generation with arbitrary timing using living electric organs of Torpedo (electric rays) which are serially integrated electrocytes converting ATP into electric energy. We developed alternative nervous systems using fluid pressure to stimulate electrocytes by a neurotransmitter, acetylcholine (Ach), and demonstrated electric generation. Maximum voltage and current were 1.5 V and 0.64 mA, respectively, with a duration time of a few seconds. We also demonstrated energy accumulation in a capacitor. The current was far larger than that using general cells other than electrocytes (~pA level). The generation ability was confirmed against repetitive cycles and also after preservation for 1 day. This is the first step toward ATP-based energy harvesting devices.

  9. An electric generator using living Torpedo electric organs controlled by fluid pressure-based alternative nervous systems

    PubMed Central

    Tanaka, Yo; Funano, Shun-ichi; Nishizawa, Yohei; Kamamichi, Norihiro; Nishinaka, Masahiro; Kitamori, Takehiko

    2016-01-01

    Direct electric power generation using biological functions have become a research focus due to their low cost and cleanliness. Unlike major approaches using glucose fuels or microbial fuel cells (MFCs), we present a generation method with intrinsically high energy conversion efficiency and generation with arbitrary timing using living electric organs of Torpedo (electric rays) which are serially integrated electrocytes converting ATP into electric energy. We developed alternative nervous systems using fluid pressure to stimulate electrocytes by a neurotransmitter, acetylcholine (Ach), and demonstrated electric generation. Maximum voltage and current were 1.5 V and 0.64 mA, respectively, with a duration time of a few seconds. We also demonstrated energy accumulation in a capacitor. The current was far larger than that using general cells other than electrocytes (~pA level). The generation ability was confirmed against repetitive cycles and also after preservation for 1 day. This is the first step toward ATP-based energy harvesting devices. PMID:27241817

  10. An electric generator using living Torpedo electric organs controlled by fluid pressure-based alternative nervous systems

    NASA Astrophysics Data System (ADS)

    Tanaka, Yo; Funano, Shun-Ichi; Nishizawa, Yohei; Kamamichi, Norihiro; Nishinaka, Masahiro; Kitamori, Takehiko

    2016-05-01

    Direct electric power generation using biological functions have become a research focus due to their low cost and cleanliness. Unlike major approaches using glucose fuels or microbial fuel cells (MFCs), we present a generation method with intrinsically high energy conversion efficiency and generation with arbitrary timing using living electric organs of Torpedo (electric rays) which are serially integrated electrocytes converting ATP into electric energy. We developed alternative nervous systems using fluid pressure to stimulate electrocytes by a neurotransmitter, acetylcholine (Ach), and demonstrated electric generation. Maximum voltage and current were 1.5 V and 0.64 mA, respectively, with a duration time of a few seconds. We also demonstrated energy accumulation in a capacitor. The current was far larger than that using general cells other than electrocytes (~pA level). The generation ability was confirmed against repetitive cycles and also after preservation for 1 day. This is the first step toward ATP-based energy harvesting devices.

  11. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  12. Putative Membrane-Bound Transporters MFSD14A and MFSD14B Are Neuronal and Affected by Nutrient Availability

    PubMed Central

    Lekholm, Emilia; Perland, Emelie; Eriksson, Mikaela M.; Hellsten, Sofie V.; Lindberg, Frida A.; Rostami, Jinar; Fredriksson, Robert

    2017-01-01

    Characterization of orphan transporters is of importance due to their involvement in cellular homeostasis but also in pharmacokinetics and pharmacodynamics. The tissue and cellular localization, as well as function, is still unknown for many of the solute carriers belonging to the major facilitator superfamily (MFS) Pfam clan. Here, we have characterized two putative novel transporters MFSD14A (HIAT1) and MFSD14B (HIATL1) in the mouse central nervous system and found protein staining throughout the adult mouse brain. Both transporters localized to neurons and MFSD14A co-localized with the Golgi marker Giantin in primary embryonic cortex cultures, while MFSD14B staining co-localized with an endoplasmic retention marker, KDEL. Based on phylogenetic clustering analyses, we predict both to have organic substrate profiles, and possible involvement in energy homeostasis. Therefore, we monitored gene regulation changes in mouse embryonic primary cultures after amino acid starvations and found both transporters to be upregulated after 3 h of starvation. Interestingly, in mice subjected to 24 h of food starvation, both transporters were downregulated in the hypothalamus, while Mfsd14a was also downregulated in the brainstem. In addition, in mice fed a high fat diet (HFD), upregulation of both transporters was seen in the striatum. Both MFSD14A and MFSD14B were intracellular neuronal membrane-bound proteins, expressed in the Golgi and Endoplasmic reticulum, affected by both starvation and HFD to varying degree in the mouse brain. PMID:28179877

  13. An investigation into membrane bound redox carriers involved in energy transduction mechanism in Brevibacterium linens DSM 20158 with unsequenced genome.

    PubMed

    Shabbiri, Khadija; Botting, Catherine H; Adnan, Ahmad; Fuszard, Matthew; Naseem, Shahid; Ahmed, Safeer; Shujaat, Shahida; Syed, Quratulain; Ahmad, Waqar

    2014-04-01

    Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158.

  14. Genome-wide identification and analysis of membrane-bound O-acyltransferase (MBOAT) gene family in plants.

    PubMed

    Wang, Peng; Wang, Zhunian; Dou, Yongchao; Zhang, Xiaoxiao; Wang, Maoyuan; Tian, Xinmin

    2013-11-01

    Membrane bound O-acyl transferase (MBOAT) family is composed of gene members encoding a variety of acyltransferase enzymes, which play important roles in plant acyl lipid metabolism. Here, we present the first genome-enabled identification and analysis of MBOAT gene models in plants. In total, we identified 136 plant MBOAT sequences from 14 plant species with complete genomes. Phylogenetic relationship analyses suggested the plant MBOAT gene models fell into four major groups, two of which likely encode enzymes of diacylglycerol acyltransferase 1 (DGAT1) and lysophospholipid acyltransferase (LPLAT), respectively, with one-three copies of paralogs present in each of the most plant species. A group of gene sequences, which are homologous to Saccharomyces cerevisiae glycerol uptake proteins (GUP), was identified in plants; copy numbers were conserved, with only one copy represented in each of the most plant species; analyses showed that residues essential for acyltransferases were more prone to be conserved than vertebrate orthologs. Among four groups, one was inferred to emerge in land plants and experience a rapid expansion in genomes of angiosperms, which suggested their important roles in adaptation of plants in lands. Sequence and phylogeny analyses indicated that genes in all four groups encode enzymes with acyltransferases. Comprehensive sequence identification of MBOAT family members and investigation into classification provide a complete picture of the MBOAT gene family in plants, and could shed light into enzymatic functions of different MBOAT genes in plants.

  15. A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

    PubMed

    Pedro, A Q; Bonifácio, M J; Queiroz, J A; Maia, C J; Passarinha, L A

    2011-11-10

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    PubMed Central

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-01-01

    Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

  17. Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions.

    PubMed

    Vagne, Quentin; Turner, Matthew S; Sens, Pierre

    2015-01-01

    The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal "corrals" and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles.

  18. Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration.

    PubMed

    Simon, Jörg; Kern, Melanie

    2008-10-01

    Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

  19. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa

    PubMed Central

    Wilton, Mike; Wong, Megan J. Q.; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D.; Lewenza, Shawn

    2016-01-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg2+ or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  20. Engineering hydrogen gas production from formate in a hyperthermophile by heterologous production of an 18-subunit membrane-bound complex.

    PubMed

    Lipscomb, Gina L; Schut, Gerrit J; Thorgersen, Michael P; Nixon, William J; Kelly, Robert M; Adams, Michael W W

    2014-01-31

    Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

  1. Aluminium and Acrylamide Disrupt Cerebellum Redox States, Cholinergic Function and Membrane-Bound ATPase in Adult Rats and Their Offspring.

    PubMed

    Ghorbel, Imen; Amara, Ibtissem Ben; Ktari, Naourez; Elwej, Awatef; Boudawara, Ons; Boudawara, Tahia; Zeghal, Najiba

    2016-12-01

    Accumulation of aluminium and acrylamide in food is a major source of human exposure. Their adverse effects are well documented, but there is no information about the health problems arising from their combined exposure. The aim of the present study was to examine the possible neurotoxic effects after co-exposure of pregnant and lactating rats to aluminium and acrylamide in order to evaluate redox state, cholinergic function and membrane-bound ATPases in the cerebellum of adult rats and their progeny. Pregnant female rats have received aluminium (50 mg/kg body weight) via drinking water and acrylamide (20 mg/kg body weight) by gavage, either individually or in combination from the 14th day of pregnancy until day 14 after delivery. Exposure to these toxicants provoked an increase in malondialdehyde (MDA) and advanced oxidation protein product (AOPP) levels and a decrease in SOD, CAT, GPx, Na(+)K(+)-ATPase, Mg(2+)-ATPase and AChE activities in the cerebellum of mothers and their suckling pups. A reduction in GSH, NPSH and vitamin C levels was also observed. These changes were confirmed by histological results. Interestingly, co-exposure to these toxicants exhibited synergism based on physical and biochemical variables in the cerebellum of mothers and their progeny.

  2. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

  3. Structure and dynamics of the membrane-bound form of the filamentous bacteriophage coat proteins by NMR spectroscopy

    SciTech Connect

    Bogusky, M.J.

    1987-01-01

    The structure and dynamics of the Pf1 and fd bacteriophage coat proteins in detergent micelles are characterized in solution by nuclear magnetic resonance spectroscopy. The coat proteins are found to exist within the bacterial inner cell membrane during viral infection and assembly. The coat proteins serve as a model system to investigate integral membrane proteins as well as the viral infection and assembly processes. The coat protein is insoluble in aqueous or organic solvents and can only be effectively solubilized in the presence of detergents that form micelles or phospholipids that form vesicles. The effective molecular weight of the detergent-micelle complex is ca. 30K daltons. Sequential assignment strategies were ineffective due to short T/sub 2s/ and severe resonance degeneracy. The backbone resonance assignments were completed by the combination of several homo- and heteronuclear correlation techniques with biosynthetic /sup 15/N labelling. 2D NOE experiments were used to locate and characterize the secondary structure of the membrane bound form of the proteins showing them to be largely helical with the hydrophobic core existing in a very stable helix.

  4. A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature.

    PubMed

    Martín, M L; Busconi, L

    2001-03-01

    Calcium-dependent protein kinases (CDPKs) are found in various subcellular localizations, which suggests that this family of serine/threonine kinases may be involved in multiple signal transduction pathways. CDPKs are believed to be involved in the response of plants to low temperatures, but the precise role in the signal transduction pathway is largely unknown. Previous reports described changes in CDPKs' mRNA levels in response to cold treatment, but whether these changes are accompanied by increases in protein level and/or kinase activities is unknown. In the present study, we identify in rice (Oryza sativa L. cv Don Juan) plants a 56-kD membrane-bound CDPK that is activated in response to cold treatment. Immunoblot analysis of the enzyme preparations from control and cold-treated plants showed that the kinase level was similar in both preparations. However, both kinase and autophosphorylating activities of the enzyme prepared from cold-treated plants were significantly higher than that obtained from control plants. The activation of the CDPK is detected after 12 to 18 h of cold treatment, which indicates that the kinase does not participate in the initial response to low temperature but in the adaptative process to adverse conditions. To our knowledge, this is the first demonstration of a CDPK that is posttranscriptionally activated in response to low temperature.

  5. Comparison of membrane-bound and soluble polyphenol oxidase in Fuji apple (Malus domestica Borkh. cv. Red Fuji).

    PubMed

    Liu, Fang; Zhao, Jin-Hong; Gan, Zhi-Lin; Ni, Yuan-Ying

    2015-04-15

    This study compared membrane-bound with soluble polyphenol oxidase (mPPO and sPPO, respectively) from Fuji apple. Purified mPPO and partially purified sPPO were used. mPPO was purified by temperature-induced phase partitioning and ion exchange chromatography. The specific activity of mPPO was 34.12× higher than that of sPPO. mPPO was more stable than sPPO at pH 5.0-8.5. Although mPPO was more easily inactivated at 25-55 °C, it is still more active than sPPO in this temperature range. The optimum substrate of mPPO was 4-methyl catechol, followed by catechol. L-cysteine had the highest inhibitory effects on mPPO followed by ascorbic acid and glutathione. Surprisingly, EDTA increased mPPO activity. The results revealed that purified mPPO is a dimer with a molecular weight of approximately 67 kDa.

  6. Protective effect of Lagenaria siceraria (Mol) against membrane-bound enzyme alterations in isoproterenol-induced cardiac damage in rats.

    PubMed

    Vijayakumar, M; Selvi, V; Krishnakumari, S

    2012-01-01

    This study was aimed at evaluating the preventive role of the ethanolic extract of Lagenaria siceraria (Mol) fruit on membrane-bound enzymes, such as sodium potassium-dependent adenosine triphosphatase (Na(+)/K(+) ATPase), calcium-dependent adenosine triphosphatase (Ca(2+) ATPase) and magnesium-dependent adenosine triphosphatase (Mg(2+) ATPase) on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Male albino Wistar rats were pretreated with the ethanolic extract of L. siceraria (Mol) fruit (125, 250 and 500 mg kg(-1) body weight) for a period of 30 days. After the treatment period, ISO (85mg kg(-1) body weight) was subcutaneously injected into rats at 24-h intervals for 2 days. ISO-induced rats showed a significant (p < 0.05) decrease in the activity of Na(+)/K(+) ATPase and an increase in the activities of Ca(2+) and Mg(2+) ATPases in the heart tissues. Pre-treatment with the ethanolic extract of L. siceraria (Mol) fruit for a period of 30 days exhibited a significant (p < 0.05) effect in ISO-induced rats. Thus, our study shows that the ethanolic extract of L. siceraria (Mol) fruit has membrane-stabilising role in ISO-induced MI in rats.

  7. The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8 functions in tolerance to bacterial blight

    PubMed Central

    Jiang, Guanghuai; Yin, Dedong; Zhao, Jiying; Chen, Honglin; Guo, Lequn; Zhu, Lihuang; Zhai, Wenxue

    2016-01-01

    Thylakoid membrane-bound ascorbate peroxidase (tAPX) is a major H2O2-scavenging enzyme. To clarify its functions in tolerance to rice bacterial blight, we produced rice lines overexpressing and suppressing tAPX (OsAPX8). The overexpressing lines exhibited increased tolerance to bacterial pathogen. The RNA interference (RNAi) lines were considerably more sensitive than the control plant. Further analysis of the H2O2 content in these transgenic plants indicated that the H2O2 accumulation of OsAPX8-overexpressing plants was considerably less than that of wild-type and RNAi plants upon challenge with bacterial pathogen. Interestingly, H2O2 was the most important factor for the serious leaf dehydration and withering of rice without major resistance genes and was not the cause of hypersensitivity. It addition, wall tightening or loosening can occur according to the level of H2O2. In addition, OsAPX8 interacted with the susceptibility protein Os8N3/Xa13, and their binding repressed the reaction of OsAPX8 in tolerance to bacterial blight. PMID:27185545

  8. Membrane-bound Dickkopf-1 in Foxp3(+) regulatory T cells suppresses T-cell-mediated autoimmune colitis.

    PubMed

    Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

    2017-10-01

    Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3(+) regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4(+) T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3(+) Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3(+) Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3(+) Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  9. Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex*

    PubMed Central

    Lipscomb, Gina L.; Schut, Gerrit J.; Thorgersen, Michael P.; Nixon, William J.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications. PMID:24318960

  10. A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16.

    PubMed

    Frielingsdorf, Stefan; Schubert, Torsten; Pohlmann, Anne; Lenz, Oliver; Friedrich, Bärbel

    2011-12-20

    The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.

  11. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells

    PubMed Central

    Moslehi, Akram; Hashemi-beni, Batool; Moslehi, Azam; Akbari, Maryam Ali

    2016-01-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs. PMID:27382350

  12. The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells.

    PubMed

    Moslehi, Akram; Hashemi-Beni, Batool; Moslehi, Azam; Akbari, Maryam Ali; Adib, Minoo

    2016-07-01

    Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs.

  13. The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8 functions in tolerance to bacterial blight.

    PubMed

    Jiang, Guanghuai; Yin, Dedong; Zhao, Jiying; Chen, Honglin; Guo, Lequn; Zhu, Lihuang; Zhai, Wenxue

    2016-05-17

    Thylakoid membrane-bound ascorbate peroxidase (tAPX) is a major H2O2-scavenging enzyme. To clarify its functions in tolerance to rice bacterial blight, we produced rice lines overexpressing and suppressing tAPX (OsAPX8). The overexpressing lines exhibited increased tolerance to bacterial pathogen. The RNA interference (RNAi) lines were considerably more sensitive than the control plant. Further analysis of the H2O2 content in these transgenic plants indicated that the H2O2 accumulation of OsAPX8-overexpressing plants was considerably less than that of wild-type and RNAi plants upon challenge with bacterial pathogen. Interestingly, H2O2 was the most important factor for the serious leaf dehydration and withering of rice without major resistance genes and was not the cause of hypersensitivity. It addition, wall tightening or loosening can occur according to the level of H2O2. In addition, OsAPX8 interacted with the susceptibility protein Os8N3/Xa13, and their binding repressed the reaction of OsAPX8 in tolerance to bacterial blight.

  14. The Novel Membrane-Bound Proteins MFSD1 and MFSD3 are Putative SLC Transporters Affected by Altered Nutrient Intake.

    PubMed

    Perland, Emelie; Hellsten, Sofie V; Lekholm, Emilia; Eriksson, Mikaela M; Arapi, Vasiliki; Fredriksson, Robert

    2017-02-01

    Membrane-bound solute carriers (SLCs) are essential as they maintain several physiological functions, such as nutrient uptake, ion transport and waste removal. The SLC family comprise about 400 transporters, and we have identified two new putative family members, major facilitator superfamily domain containing 1 (MFSD1) and 3 (MFSD3). They cluster phylogenetically with SLCs of MFS type, and both proteins are conserved in chordates, while MFSD1 is also found in fruit fly. Based on homology modelling, we predict 12 transmembrane regions, a common feature for MFS transporters. The genes are expressed in abundance in mice, with specific protein staining along the plasma membrane in neurons. Depriving mouse embryonic primary cortex cells of amino acids resulted in upregulation of Mfsd1, whereas Mfsd3 is unaltered. Furthermore, in vivo, Mfsd1 and Mfsd3 are downregulated in anterior brain sections in mice subjected to starvation, while upregulated specifically in brainstem. Mfsd3 is also attenuated in cerebellum after starvation. In mice raised on high-fat diet, Mfsd1 was specifically downregulated in brainstem and hypothalamus, while Mfsd3 was reduced consistently throughout the brain.

  15. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris.

    PubMed

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-02-15

    Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens.

  16. Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions

    PubMed Central

    Vagne, Quentin; Turner, Matthew S.; Sens, Pierre

    2015-01-01

    The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal “corrals” and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

  17. Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

    PubMed Central

    Jurat-Fuentes, Juan Luis; Karumbaiah, Lohitash; Jakka, Siva Rama Krishna; Ning, Changming; Liu, Chenxi; Wu, Kongming; Jackson, Jerreme; Gould, Fred; Blanco, Carlos; Portilla, Maribel; Perera, Omaththage; Adang, Michael

    2011-01-01

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests. PMID:21390253

  18. The Mössbauer Parameters of the Proximal Cluster of Membrane-Bound Hydrogenase Revisited: A Density Functional Theory Study

    PubMed Central

    2015-01-01

    An unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, 57Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S]3+ reduced-state proximal cluster. Similar conflicting assignments are also resolved for the [4Fe-3S]5+ superoxidized state with particular regard to spin-coupling in the broken-symmetry DFT calculations. Calculated 57Fe hyperfine coupling (HFC) tensors expose discrepancies in the experimental set of HFC tensors and substantiate the need for additional experimental work on the magnetic properties of the MBH proximal cluster in its reduced and superoxidized redox states. PMID:26598030

  19. Torpedo electromotor system development: a quantitative analysis of synaptogenesis.

    PubMed

    Fox, G Q; Kötting, D

    1984-04-10

    Synaptogenesis in the electric organ of Torpedo marmorato has been studied quantitatively at the ultrastructural level of observation. In addition to establishing the normal developmental time course for this event we were interested in determining whether a gradient of synaptogenesis might be present because the electric organ produces several morphologically recognizable spatiotemporal gradients during its early ontogeny. These gradients genesis of electrocyte columns, both gradients of which are operative for periods of weeks. No gradient of synaptogenesis was found, indicating this to be a synchronous process. The idea is advanced that synaptogenesis in the electric organ is modulated by extrinsic influences, many of which may originate from the target electrocytes which, by this time, have become synchronized in their development.

  20. [Bacterial expression of water-soluble domain of Lynx1, endogenic neuromodulator of human nicotinic acetylcholine receptors].

    PubMed

    Shulepko, M A; Liukmanova, E N; Kasheverov, I E; Dolgikh, D A; Tsetlin, V I; Kirpichnikov, M P

    2011-01-01

    Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.

  1. Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects.

    PubMed

    Breer, H; Kleene, R; Hinz, G

    1985-12-01

    The nicotinic acetylcholine receptor as probed by alpha-bungarotoxin binding has been isolated from detergent-solubilized ganglionic membrane preparations from the insect, Locusta migratoria. The isolation and characterization of the receptor protein was achieved by preparation of membrane fragments, extraction by sodium deoxycholate, centrifugation on sucrose density gradient, affinity chromatography, gel electrophoresis, and immunoblotting. The purified receptor protein migrated as a single band on polyacrylamide when native (Mr = 250,000 to 300,000) but also under denaturing conditions (Mr = 65,000) and cross-reacted with some monoclonal antibodies against the Torpedo receptor. In immunohistochemical approaches using polyclonal antibodies the acetylcholine receptor antigenic sites could topochemically be identified at very distinct zones in the neuropil of locust ganglia. The results suggest that the acetylcholine receptor in the central nervous system of insects represents an oligomeric complex composed of four identical or very similar subunits and thus may represent a prototype of the recently proposed homo-oligomeric ancestral acetylcholine receptor.

  2. Subunit structure of the acetylcholine receptor from Electrophorus electricus.

    PubMed Central

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A

    1982-01-01

    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR. Images PMID:6959131

  3. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  4. Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

    PubMed

    Araujo, Carolina Morais; Hermidorff, Milla Marques; Amancio, Gabriela de Cassia Sousa; Lemos, Denise da Silveira; Silva, Marcelo Estáquio; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

    2016-10-01

    Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

  5. Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

    PubMed

    Li, Mei-Hui

    2012-04-01

    Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period. Copyright © 2012 SETAC.

  6. Niche anchorage and signaling through membrane-bound Kit-ligand/c-kit receptor are kinase independent and imatinib insensitive.

    PubMed

    Tabone-Eglinger, Séverine; Calderin-Sollet, Zuleika; Pinon, Perrine; Aebischer, Nicole; Wehrle-Haller, Monique; Jacquier, Marie-Claude; Boettiger, David; Wehrle-Haller, Bernhard

    2014-10-01

    Kit ligand (KitL) and its tyrosine kinase receptor c-kit are critical for germ cells, melanocytes, mastocytes, and hematopoietic stem cells. Alternative splicing of KitL generates membrane-bound KitL (mb-KitL) or soluble KitL, providing survival or cell migration, respectively. Here we analyzed whether c-kit can function both as an adhesion and signaling receptor to mb-KitL presented by the environmental niche. At contacts between fibroblasts and MC/9 mast cells, mb-KitL, and c-kit formed ligand/receptor clusters that formed stable complexes, which resisted dissociation by c-kit blocking mAbs and provided cell anchorage under physiological shear stresses. Clusters recruited tyrosine-phosphorylated proteins and induced spatially restricted F-actin polymerization. Mutational analysis of c-kit demonstrated kinase-independent mb-KitL/c-kit clustering, anchorage, F-actin polymerization, and Tyr567-dependent cluster phosphorylation. Kinase inhibition of c-kit by imatinib reduced cluster coalescence, but allowed cluster phosphorylation and F-actin polymerization, which required PI3K recruitment and a newly identified juxtamembrane residue. Synergies between integrin and c-kit-mediated spreading and adhesion of MC/9 cells were studied in vitro on immobilized-KitL/fibronectin surfaces. While c-kit blocking antibodies prevented spreading, imatinib blocked spreading induced by soluble- but not immobilized KitL. Thus, "mechanical" activation of c-kit provides signaling, niche-anchorage, and synergies with integrin-mediated adhesion, which is independent of kinase function and resistant to c-kit kinase inhibitors.-

  7. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    DOE PAGES

    Bansal, Sunil; Durrett, Timothy P.

    2016-11-08

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

  8. Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2.

    PubMed

    Chang, Mi-Ra; Lee, Woong-Hee; Choi, Jin-Wha; Park, Sun-Ok; Paik, Sang-Gi; Kim, Young Sang

    2005-06-30

    Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8(+) T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8(+) T cell-depleted mice, but not in CD4(+) T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8(+) T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4(+) and CD8(+) T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8(+) T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.

  9. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.

    PubMed

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw(-1) h(-1) was observed at a dilution rate of 0.31 h(-1). We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h(-1), the SHPR was 36.2 mmol H2 g-dcw(-1) h(-1), corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h(-1) or 1.07 h(-1) resulted in a SHPR of 120 mmol H2 g-dcw(-1) h(-1), which is one of the highest production rates observed in microbial fermentation.

  10. Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

    PubMed Central

    Frescas, David; Roux, Christelle M.; Aygun-Sunar, Semra; Gleiberman, Anatoli S.; Krasnov, Peter; Kurnasov, Oleg V.; Strom, Evguenia; Virtuoso, Lauren P.; Wrobel, Michelle; Osterman, Andrei L.; Antoch, Marina P.; Mett, Vadim; Chernova, Olga B.; Gudkov, Andrei V.

    2017-01-01

    Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation. PMID:28193858

  11. Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

    PubMed Central

    Osman, T A; Buck, K W

    1996-01-01

    A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme. PMID:8709249

  12. Oxygen reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases.

    PubMed

    Lamrabet, O; Pieulle, L; Aubert, C; Mouhamar, F; Stocker, P; Dolla, A; Brasseur, G

    2011-09-01

    Although Desulfovibrio vulgaris Hildenborough (DvH) is a strictly anaerobic bacterium, it is able to consume oxygen in different cellular compartments, including extensive periplasmic O₂ reduction with hydrogen as electron donor. The genome of DvH revealed the presence of cydAB and cox genes, encoding a quinol oxidase bd and a cytochrome c oxidase, respectively. In the membranes of DvH, we detected both quinol oxygen reductase [inhibited by heptyl-hydroxyquinoline-N-oxide (HQNO)] and cytochrome c oxidase activities. Spectral and HPLC data for the membrane fraction revealed the presence of o-, b- and d-type haems, in addition to a majority of c-type haems, but no a-type haem, in agreement with carbon monoxide-binding analysis. The cytochrome c oxidase is thus of the cc(o/b)o₃ type, a type not previously described. The monohaem cytochrome c₅₅₃ is an electron donor to the cytochrome c oxidase; its encoding gene is located upstream of the cox operon and is 50-fold more transcribed than coxI encoding the cytochrome c oxidase subunit I. Even when DvH is grown under anaerobic conditions in lactate/sulfate medium, the two terminal oxidase-encoding genes are expressed. Furthermore, the quinol oxidase bd-encoding genes are more highly expressed than the cox genes. The cox operon exhibits an atypical genomic organization, with the gene coxII located downstream of coxIV. The occurrence of these membrane-bound oxygen reductases in other strictly anaerobic Deltaproteobacteria is discussed.

  13. Characteristics of Membrane-Bound and Free Hepatic Ribosomes from Insulin-Deficient Rats I. ACUTE EXPERIMENTAL DIABETES MELLITUS

    PubMed Central

    Peterson, Daniel T.; Alford, Frank P.; Reaven, Eve P.; Ueyama, Irene; Reaven, Gerald M.

    1973-01-01

    Membrane-bound and free ribosomes were prepared by discontinuous density gradient centrifugation from livers of rats 2-3 days after receiving alloxan (75 mg/kg) or streptozotocin (100 mg/kg). Hepatocytes from these animals were also examined by electron microscopy and subjected to quantitative morphometric analysis. The results indicated that the two populations of hepatic ribosomes respond differently to acute insulin deficiency. There was an overall reduction (P < 0.001) in total number of bound ribosomes per volume cytoplasm: the remaining bound ribosomes underwent a shift to smaller-sized ribosomal messenger RNA (mRNA) aggregates (P < 0.02); and the proteinsynthetic activity of these bound ribosomes was less than normal (P < 0.02) when protein synthesis was directed by endogenous mRNA. However, there was no difference between bound ribosomes from livers of normal and diabetic rats when protein synthesis was directed by polyuridylic acid. In contrast, free ribosomes were unchanged in number and degree of ribosomal mRNA aggregation, but displayed a significantly increased rate of in vitro protein synthesis (P < 0.01) as compared to normal controls. This increased protein-synthetic activity occurred when amino acid incorporation was directed by endogenous mRNA or polyuridylic acid. These changes in structure and function of bound and free hepatic ribosomes were prevented by the concomitant administration of insulin. The decrease in protein-synthetic activity of bound hepatic ribosomes from acutely diabetic rats seems to be secondary to marked disruption and disaggregation of the rough endoplasmic reticulum (RER) with production of smaller ribosomal mRNA aggregates which incorporate less amino acids into protein. Increased protein synthetic activity of free ribosome appears to be related to the ability of these ribosomes to copy mRNA more efficiently. Images PMID:4270772

  14. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    PubMed Central

    Bansal, Sunil; Durrett, Timothy P.

    2016-01-01

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

  15. Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

    PubMed Central

    Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

    2014-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

  16. Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production

    PubMed Central

    Kanai, Tamotsu; Simons, Jan-Robert; Tsukamoto, Ryohei; Nakajima, Akihito; Omori, Yoshiyuki; Matsuoka, Ryoji; Beppu, Haruki; Imanaka, Tadayuki; Atomi, Haruyuki

    2015-01-01

    The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw−1 h−1 was observed at a dilution rate of 0.31 h−1. We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h−1, the SHPR was 36.2 mmol H2 g-dcw−1 h−1, corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h−1 or 1.07 h−1 resulted in a SHPR of 120 mmol H2 g-dcw−1 h−1, which is one of the highest production rates observed in microbial fermentation. PMID:26379632

  17. Depression of membrane-bound Na sup + -K sup + -ATPase activity induced by free radicals and by ischemia of kidney

    SciTech Connect

    Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. )

    1988-02-01

    A partially purified, membrane-bound Na{sup +}-K{sup +}-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H{sub 2}O{sub 2} for either 15 or 30 min at 37{degree}C. The activity of ouabain-sensitive Na{sup +}-K{sup +}-ATPase was reduced proportionally to the concentration of H{sub 2}O{sub 2} and the duration of incubation. There were decreases in SH contents and turnover rates of the Na{sup +}-K{sup +}-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na{sup +}K{sup +}-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4{degree}C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na{sup +}-K{sup +}-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney.

  18. A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

    PubMed Central

    Bécamel, Carine; Galéotti, Nathalie; Poncet, Joël; Jouin, Patrick; Dumuis, Aline; Bockaert, Joël

    2002-01-01

    There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). PMID:12734563

  19. Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

    PubMed Central

    Bhushan, A; McNamee, M G

    1993-01-01

    Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy. PMID:8471723

  20. Homogeneous purification and characterization of LePGT1--a membrane-bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon.

    PubMed

    Ohara, Kazuaki; Mito, Koji; Yazaki, Kazufumi

    2013-06-01

    Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.

  1. Protective effect of fish oil on changes in the activities of membrane-bound ATPases and mineral status in experimentally induced myocardial infarction in Wistar rats.

    PubMed

    Padma, Viswanadha Vijaya; Devi, Chennam Srinivasulu Shyamala; Kalaiselvi, Palaniswamy

    2010-12-01

    The present study evaluated the protective effect of fish oil in isoproterenol-induced myocardial infarction in rats. The results of the present study indicate that the IPH administration decreases the activities of membrane-bound ATPases compared to control animals. Fish oil pretreatment brought about significant increase in the activity of these membrane-bound ATPases in IPH (isoproterenol hydrochloride)-treated animals. Significant increase in serum potassium level with concomitant decrease in the values of sodium, magnesium, and calcium were observed in IPH-treated rats compared to control rats, fish oil pretreatment reversed these changes to near normal. Significant elevation of sodium and calcium levels with concomitant decrease in the levels of potassium and magnesium were observed in the myocardial tissue of IPH-administered rats compared to control rats, fish oil pretreatment followed by IPH administration brought these levels to near normal. The levels of lipid peroxidation (LPO) in both serum and tissue were increased in IPH-treated rats compared with control rats, whereas pretreatment with fish oil in IPH-treated rats maintained near-normal LPO levels. The results of the present study reveals that the pretreatment of fish maintains the activities of membrane-bound ATPases and the mineral levels at near normal by the inhibition of lipid peroxidation.

  2. Generation of membrane-bound catechol-O-methyl transferase deficient mice with disctinct sex dependent behavioral phenotype.

    PubMed

    Tammimaki, A; Aonurm-Helm, A; Zhang, F P; Poutanen, M; Duran-Torres, G; Garcia-Horsman, A; Mannisto, P T

    2016-12-01

    Catechol-O-methyltransferase (COMT) has two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-MT), anchored to intracellular membranes. COMT is involved in the O-methylation of L-DOPA, dopamine and other catechols. The exact role of MB-COMT is still mostly unclear. We wanted to create a novel genetically modified mouse model that specifically lacks MB-COMT activity and to study their behavioral phenotype. MB-COMT knock-in mutant mice were generated by introducing two point mutations in exon 2 of the Comt gene (ATGCTG->GAGCTC disabling the function of the P2 promoter and allowing only the P1-regulated S-COMT transcription. The first mutation changes methionine to glutamic acid whereas the second one does not affect coding. The expression of the two COMT isoforms, total COMT activity in several areas of the brain and peripheral tissues and extracellular dopamine concentrations after L-DOPA (10 mg/kg) and carbidopa (30 mg/kg) subcutaneous administration were assessed. A battery of behavioral tests was performed to compare MB-COMT deficient mice and their wild type littermates of both sexes. MB-COMT deficient mice were seemingly normal, bred usually and had unaltered COMT activity in the brain and periphery despite a complete lack of the MB-COMT protein. MB-COMT deficient male mice showed higher extracellular dopamine levels than their wild-type littermates in the striatum, but not in the mPFC. In addition, the MB-COMT deficient male mice exhibited a distinct endophenotype characterized by schizophrenia-related behaviors like aggressive behavior and reduced prepulse inhibition. They also had prolonged immobility in the tail suspension test. Both sexes were sensitized to acute pain and had normal motor activity but disturbed short-term memory. Hence the behavioral phenotype was not limited to schizophrenia-related endophenotype and some behavioural findings were not sex-dependent. Our findings indicate that MB-COMT is critical for

  3. Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes

    PubMed Central

    1994-01-01

    Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together

  4. THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA

    PubMed Central

    Sheridan, Michael N.

    1965-01-01

    The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque. PMID:14286287

  5. Probing Protein Packing Surrounding the Residues In and Flanking the Nicotinic Acetylcholine Receptor M2M3 Loop

    PubMed Central

    Wiltfong, Roger Ernest; Jansen, Michaela

    2009-01-01

    Nicotinic Acetylcholine Receptors (nAChR) are cation-selective, ligand-gated ion channels of the Cys-loop gene superfamily. The recent crystal structure of a bacterial homologue from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of acetylcholine-binding protein (AChBP) and of the electronmicroscopy derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method (SCAM). The M2 20′ to 27′ residues were highly water accessible and the variation in reaction rates were consistent with this region being α-helical. At all positions tested, the presence of ACh changed MTSEA modification rates by less than 10-fold. In the presence of ACh, reaction rates for residues in the last extracellular α-helical turn of M2 and in the M2M3 loop increased, whereas rates in M2's penultimate α-helical turn decreased. Only 3 out of 8 M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with it's location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys loop receptors. PMID:19211870

  6. Morphogenetic roles of acetylcholine.

    PubMed Central

    Lauder, J M; Schambra, U B

    1999-01-01

    In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences. PMID:10229708

  7. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.

    PubMed

    Kasheverov, Igor E; Utkin, Yuri N; Tsetlin, Victor I

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

  8. Association of social defeat stress-induced anhedonia-like symptoms with mGluR1-dependent decrease in membrane-bound AMPA-GluR1 in the mouse ventral midbrain.

    PubMed

    Yashiro, Sayori; Seki, Kenjiro

    2017-07-01

    Anhedonia is a core symptom of social defeat stress (SDS)-induced depression associated with the reward system. We previously reported that decreased membrane-bound AMPA-GluR1 in the reward system is associated with lipopolysaccharide-induced anhedonia-like symptoms. Since group I metabotropic glutamate receptor (mGluR) activation reduces the surface density of GluR1, we examined whether group I mGluR-dependent decrease in membrane-bound GluR1 in the reward system is involved in SDS-induced anhedonia-like symptoms. Mice exposed to SDS for 4 consecutive days had markedly decreased membrane-bound GluR1 and GluR2 in the prefrontal cortex (PFC) and membrane-bound GluR1 in the ventral midbrain (VM) along with lower sucrose preference (SP). Intra-PFC injection of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μmol) demonstrated decrease in membrane-bound GluR1 and GluR2 in the PFC 2 and 24 h and membrane-bound GluR1 in the VM 24 h after injection. Moreover, intra-PFC injection of DHPG decreased SP only in the second 24-h (24-48 h) period. Conversely, intra-VM injection of DHPG decreased SP in both the first and second 24-h period and decreased membrane-bound GluR1 in the VM 2 and 24 h after injection. Pre-treatment with the mGluR1 antagonist JNJ16259685 (30 mg/kg, subcutaneous) prevented SDS-decreased SP and membrane-bound GluR1 in the VM. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP; 10 mg/kg, subcutaneous) prevented SDS-induced decrease in membrane-bound GluR1 and GluR2 in the PFC, whereas MPEP did not affect SDS-induced decrease in SP and membrane-bound GluR1 in the VM. These results suggest that mGluR1-mediated decrease in membrane-bound GluR1 in VM is involved in SDS-induced anhedonia-like symptoms.

  9. Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

    PubMed

    Sikorav, J L; Grassi, J; Bon, S

    1984-12-17

    We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

  10. Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

    PubMed Central

    Behling, R W; Yamane, T; Navon, G; Sammon, M J; Jelinski, L W

    1988-01-01

    A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state. Images FIGURE 1 PMID:3395661

  11. Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

    SciTech Connect

    Wadsworth, S.C.; Rosenthal, L.S.; Kammermeyer, K.L.; Potter, M.B.; Nelson, D.J.

    1988-02-01

    The authors isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobasae poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

  12. [Influence of UV-light on erythrocyte membrane structure and catalytic behaviour of membrane acetylcholine esterase].

    PubMed

    Volotovskiĭ, I D; Sheĭko, L M; Konev, S V

    1976-01-01

    UV-light is shown to induce the structural transitions in the erythrocyte membrane described by S-shape curves in plots of the structural response versus the irradiation dose. In contrast to the free acetylcholine esterase (AChE) UV-light acts on the membrane enzyme as a mixed inhibitor (simultaneous change in Vmax and Km). The modification of the environment structure of residual enzyme is suggested to be the main reason of this phenomenon. The effect is under the control of membrane integrity and disappears after its desintegration. Membrane AChE treated ultrasonically both prior to and after irradiation is inactivated without a Km change. The data obtained show the influence of erythrocyte membrane structure on the catalytic behaviour of membrane-bound AChE.

  13. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    SciTech Connect

    Gainer, M.W.

    1985-01-01

    Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3(3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, (/sup 3/H)quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold.

  14. Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen.

    PubMed

    Weiner, Lev; Roth, Esther; Silman, Israel

    2011-01-01

    The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts. © 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.

  15. Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo.

    PubMed

    Fiedler, W; Walker, J H

    1985-07-01

    Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.

  16. Torpedo Maculopathy in a 6-Month-Old Infant: Early Clinical and Optical Coherence Tomography Findings.

    PubMed

    Kara, Caner; Petriçli, İkbal Seza

    2017-08-24

    A 6-month-old male infant presented for routine ophthalmologic examination. Indirect ophthalmoscopy revealed a flat, oval, hypopigmented lesion located in the temporal macula in the right eye with the tip pointing toward the fovea, which was compatible with torpedo maculopathy. Optical coherence tomography (OCT) was performed at the time of diagnosis. OCT scans of the lesion revealed slight retinal pigment epithelium hyperreflectivity. This case serves as the earliest OCT finding of the youngest patient diagnosed as having torpedo maculopathy in the literature. [J Pediatr Ophthalmol Strabismus. 2017;54:e54-e57.]. Copyright 2017, SLACK Incorporated.

  17. Enhancement of cell growth and glycolic acid production by overexpression of membrane-bound alcohol dehydrogenase in Gluconobacter oxydans DSM 2003.

    PubMed

    Zhang, Huan; Shi, Lulu; Mao, Xinlei; Lin, Jinping; Wei, Dongzhi

    2016-11-10

    Membrane-bound alcohol dehydrogenase (mADH) was overexpressed in Gluconobacter oxydans DSM 2003, and the effects on cell growth and glycolic acid production were investigated. The transcription levels of two terminal ubiquinol oxidases (bo3 and bd) in the respiratory chain of the engineered strain G. oxydans-adhABS were up-regulated by 13.4- and 3.8-fold, respectively, which effectively enhanced the oxygen uptake rate, resulting in higher resistance to acid. The cell biomass of G. oxydans-adhABS could increase by 26%-33% when cultivated in a 7L bioreactor. The activities of other major membrane-bound dehydrogenases were also increased to some extent, particularly membrane-bound aldehyde dehydrogenase (mALDH), which is involved in the catalytic oxidation of aldehydes to the corresponding acids and was 1.26-fold higher. Relying on the advantages of the above, G. oxydans-adhABS could produce 73.3gl(-1) glycolic acid after 45h of bioconversion with resting cells, with a molar yield 93.5% and a space-time yield of 1.63gl(-1)h(-1). Glycolic acid production could be further improved by fed-batch fermentation. After 45h of culture, 113.8gl(-1) glycolic acid was accumulated, with a molar yield of 92.9% and a space-time yield of 2.53gl(-1)h(-1), which is the highest reported glycolic acid yield to date. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

    PubMed Central

    Tan, Shu-Tang; Xue, Hong-Wei

    2016-01-01

    Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

  19. Analysis of local conformation of membrane-bound and polycrystalline peptides by two-dimensional slow-spinning rotor-synchronized MAS exchange spectroscopy.

    PubMed

    Gabrys, Charles M; Yang, Jun; Weliky, David P

    2003-05-01

    2D slow-spinning, rotor-synchronized MAS exchange spectroscopy (SSRS-MASE) was applied to study local secondary structure of three structurally different peptides, two of which were membrane-bound. Each peptide was (13)C carbonyl labeled at two adjacent residues in the peptide backbone. In general, this methodology is attractive for membrane-bound peptides because of its lenient spinning, decoupling, and RF homogeneity requirements. For a single set of raw SSRS-MASE data, two linearly independent methods exist for obtaining a 2D spectrum and each spectrum can be fit to obtain conformational constraints. An approach is described for combining the results of these two fits and this method is shown to work for spectra with both resolved and unresolved labeled site resonances. A spectrum is often fit well to a few different conformations which have somewhat different values of the fitting parameter chi(2). A simple statistical theory is developed which relates the deltachi(2) difference between a local minimum and the global minimum chi(2) to the likelihood that the local minimum conformation is the correct structure. Because uncertainty in the simulated data can also contribute to the overall fitting uncertainty, an empirical method is described for incorporating the simulation uncertainty into the deltachi(2) analysis. These data analysis methods were tested on polycrystalline Ala-Gly-Gly and then applied to the membrane-bound melittin and HIV-1 fusion peptides. Melittin gave a best-fit alpha helical structure at Ala-4 while the fusion peptide gave a good-fit beta strand structure at Phe-8. The melittin analysis is in agreement with the known overall structure of this peptide.

  20. Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

    PubMed Central

    Ashcom, J D; Stiles, B G

    1997-01-01

    The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies. PMID:9359860

  1. Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor*

    PubMed Central

    Utkin, Yuri N.; Weise, Christoph; Kasheverov, Igor E.; Andreeva, Tatyana V.; Kryukova, Elena V.; Zhmak, Maxim N.; Starkov, Vladislav G.; Hoang, Ngoc Anh; Bertrand, Daniel; Ramerstorfer, Joachim; Sieghart, Werner; Thompson, Andrew J.; Lummis, Sarah C. R.; Tsetlin, Victor I.

    2012-01-01

    Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges. PMID:22613724

  2. Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor.

    PubMed

    Utkin, Yuri N; Weise, Christoph; Kasheverov, Igor E; Andreeva, Tatyana V; Kryukova, Elena V; Zhmak, Maxim N; Starkov, Vladislav G; Hoang, Ngoc Anh; Bertrand, Daniel; Ramerstorfer, Joachim; Sieghart, Werner; Thompson, Andrew J; Lummis, Sarah C R; Tsetlin, Victor I

    2012-08-03

    Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

  3. Characterization and inactivation of the membrane-bound polyol dehydrogenase in Gluconobacter oxydans DSM 7145 reveals a role in meso-erythritol oxidation.

    PubMed

    Voss, Jörn; Ehrenreich, Armin; Liebl, Wolfgang

    2010-06-01

    The growth of Gluconobacter oxydans DSM 7145 on meso-erythritol is characterized by two stages: in the first stage, meso-erythritol is oxidized almost stoichiometrically to L-erythrulose according to the Bertrand-Hudson rule. The second phase is distinguished from the first phase by a global metabolic change from membrane-bound meso-erythritol oxidation to L-erythrulose assimilation with concomitant accumulation of acetic acid. The membrane-associated erythritol-oxidizing enzyme was found to be encoded by a gene homologous to sldA known from other species of acetic acid bacteria. Disruption of this gene in the genome of G. oxydans DSM 7145 revealed that the membrane-bound polyol dehydrogenase not only oxidizes meso-erythritol but also has a broader substrate spectrum which includes C3-C6 polyols and D-gluconate and supports growth on these substrates. Cultivation of G. oxydans DSM 7145 on different substrates indicated that expression of the polyol dehydrogenase was not regulated, implying that the production of biomass of G. oxydans to be used as whole-cell biocatalysts in the biotechnological conversion of meso-erythritol to L-erythrulose, which is used as a tanning agent in the cosmetics industry, can be conveniently carried out with glucose as the growth substrate.

  4. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  5. First evidence of a membrane-bound, tyramine and beta-phenylethylamine producing, tyrosine decarboxylase in Enterococcus faecalis: a two-dimensional electrophoresis proteomic study.

    PubMed

    Pessione, Enrica; Pessione, Alessandro; Lamberti, Cristina; Coïsson, Daniel Jean; Riedel, Kathrin; Mazzoli, Roberto; Bonetta, Silvia; Eberl, Leo; Giunta, Carlo

    2009-05-01

    The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence.

  6. Efficacy of Sargassum polycystum (Phaeophyceae) sulphated polysaccharide against paracetamol-induced DNA fragmentation and modulation of membrane-bound phosphatases during toxic hepatitis.

    PubMed

    Raghavendran, H B; Sathivel, A; Yogeeta, R S S K; Devaki, T

    2007-03-01

    1. The aim of the present study was to assess the protective effect of Sargassum polycystum (sulphated polysaccharide) extract against paracetamol-induced DNA strand breaks and modulation of membrane-bound phosphatases, protein thiols and inorganic cations during toxic hepatitis. 2. Seaweed extract (200 mg/kg per day for 21 days) was administered to male Wistar rats against paracetamol challenge. Serum and liver tissues were used to assess levels of ATPase, protein thiols and inorganic cations using atomic absorption spectroscopy. The fragmentation of DNA was assessed by agarose gel electrophoresis. 3. Paracetamol induced intracellular stress, accompanied by changes in the structural and functional characteristics of liver cell membranes, which affected DNA integrity, membrane-bound ATPase and inorganic cations homeostasis. Rats intoxicated with paracetamol (800 mg/kg, i.p.) showed significant impairment in activities of total ATPase, Mg2+-ATPase, Ca+-ATPase and Na+/K+-ATPase, with concomitant changes in the levels of tissue protein thiols and inorganic cations, such as Na+, K+ and Ca2+. These changes were prevented in animals pretreated with S. polycystum extract, which indicates that S. polycystum supplementation could exert some protective effect against paracetamol-induced toxic hepatitis in rats. 4. The protective effect of the seaweed extract may be due to the presence of sulphated compounds that have free radical-scavenging activity.

  7. Protective effect of Premna tomentosa (L. Verbenaceae) extract on membrane-bound phosphatases and inorganic cations transport in acetaminophen-induced hepatotoxicity rats.

    PubMed

    Devi, K Pandima; Sreepriya, M; Balakrishna, K; Devaki, T

    2004-08-01

    Hepatic injury elicits intracellular stress that leads to peroxidation of membrane lipids accompanied by alteration of structural and functional characteristics of membrane, which affect the activities of membrane-bound ATPases. The present study appraised the membrane protective effect of Premna tomentosa, a hepatoprotective drug used in Indian traditional medicine. Wistar strain rats were pre-treated with Premna tomentosa extract (750 mg/kg, orally) for 15 days, 24 h prior to administration of acetaminophen (640 mg/kg, orally). During acetaminophen intoxication, the levels of membrane-bound enzymes were significantly decreased, total ATPase (1.63-fold), Mg(2+)ATPase (1.9-fold), Ca(2+)ATPase (1.33-fold) and Na(+)K(+)ATPase (1.73-fold) which was accompanied by changes in the levels of inorganic cations N+, K+ and Ca2+. These alterations were prevented by Premna tomentosa extract pre-treatment, which shows that Premna tomentosa supplementation could exert a beneficial effect against liver injury-induced membrane damage. The potential of the plant might be credited to the presence of antioxidant compound limonene in the plant.

  8. Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

    PubMed Central

    Rath, P; Bousché, O; Merrill, A R; Cramer, W A; Rothschild, K J

    1991-01-01

    The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin. PMID:1710937

  9. Identification of a membrane-bound, glycol-stimulated phospholipase A sub 2 located in the secretory granules of the adrenal medulla

    SciTech Connect

    Hildebrandt, E.; Albanesi, J.P. )

    1991-01-01

    Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca{sup 2+}-stimulated phospholipase A{sub 2} (PLA{sub 2}) activity when assayed at pH 9.0 with phosphatidylcholine containing an ({sup 14}C)-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA{sub 2} activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycole, or poly(ethylene glycol). This glycol-stimulated PLA{sub 2} activity codistributed with membrane-bound dopamine {beta}-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA{sub 2} of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different {sup 14}C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethhanolamine, and distearoylphosphatidylcholine was not hydrolyzed.

  10. The Autocrine Mitogenic Loop of the Ciliate Euplotes raikovi: The Pheromone Membrane-bound Forms Are the Cell Binding Sites and Potential Signaling Receptors of Soluble Pheromones

    PubMed Central

    Ortenzi, Claudio; Alimenti, Claudio; Vallesi, Adriana; Di Pretoro, Barbara; Terza, Antonietta La; Luporini, Pierangelo

    2000-01-01

    Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding. PMID:10749941

  11. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  12. Membrane-bound ICAM-1 contributes to the onset of proinvasive tumor stroma by controlling acto-myosin contractility in carcinoma-associated fibroblasts

    PubMed Central

    Bonan, Stephanie; Albrengues, Jean; Grasset, Eloise; Kuzet, Sanya-Eduarda; Nottet, Nicolas; Bourget, Isabelle; Bertero, Thomas; Mari, Bernard; Meneguzzi, Guerrino; Gaggioli, Cedric

    2017-01-01

    Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination. PMID:27901489

  13. A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism.

    PubMed

    Yang, Xue-Peng; Wei, Liu-Jing; Ye, Jian-Bin; Yin, Bo; Wei, Dong-Zhi

    2008-09-15

    A membrane-bound pyrroloquinoline quinine (PQQ)-dependent D-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of D-sorbitol to L-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by D-sorbitol), while products are released in sequence (first L-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for D-sorbitol, in addition to PQQ-binding and Mg(2+)-binding sites in the catalytic center.

  14. Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

    PubMed Central

    Mallikarjuna, Nimgampalle; Praveen, Kukkarasapalli; Yellamma, Kuna

    2016-01-01

    Introduction: Alzheimer’s disease (AD) is a neurodegenerative disorder, clinically characterized by memory dysfunction and progressive loss of cognition. No curative therapeutic or drug is available for the complete cure of this disease. The present study was aimed to evaluate the efficacy of Lactobacillus plantarum MTCC1325 in ATPases activity in the selected brain regions of rats induced with Alzheimer’s. Methods: For the study, 48 healthy Wistar rats were divided into four groups: group I as control group, group II as AD model (AD induced by intraperitoneal injection of D-Galactose, 120 mg/kg body weight for 6 weeks), group III as normal control rats which were orally administered only with L. plantarum MTCC1325 for 60 days, and group IV where the AD-induced rats simultaneously received oral treatment of L. plantarum MTCC1325 (10ml/kg body weight, 12×108 CFU/mL) for 60 days. The well known membrane bound transport enzymes including Na+, K+-ATPases, Ca2+-ATPases, and Mg2+-ATPases were assayed in the selected brain regions of hippocampus and cerebral cortex in all four groups of rats at selected time intervals. Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20%) in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration. PMID:28265536

  15. Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors

    PubMed Central

    Mika, Angela; Boenisch, Marike Johanne; Hopff, David; Lüthje, Sabine

    2010-01-01

    Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H2O2 treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses. PMID:20032108

  16. The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

    PubMed

    Food, Michael R; Sekyere, Eric O; Richardson, Des R

    2002-09-01

    Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.

  17. Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation.

    PubMed

    Ramel, F; Amrani, A; Pieulle, L; Lamrabet, O; Voordouw, G; Seddiki, N; Brèthes, D; Company, M; Dolla, A; Brasseur, G

    2013-12-01

    Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

  18. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

    PubMed Central

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-01-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  19. Synergistic salubrious effect of ferulic acid and ascorbic acid on membrane-bound phosphatases and lysosomal hydrolases during experimental myocardial infarction in rats.

    PubMed

    Yogeeta, Surinder Kumar; Gnanapragasam, Arunachalam; Senthilkumar, Subramanian; Subhashini, Rajakannu; Devaki, Thiruvengadam

    2006-12-23

    Altered membrane integrity has been suggested as a major factor in the development of cellular injury during myocardial necrosis. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on lysosomal hydrolases and membrane-bound phosphatases during isoproterenol (ISO) induced myocardial necrosis in rats. Induction of rats with 1SO (150 mg/kg b.wt, i.p.) for 2 days resulted in a significant increase in the activities of lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase and cathepsin-D) in the heart and serum. A significant increase in plasma lactate level, cardiac levels of sodium, calcium and a decrease in cardiac level of potassium was also observed, which was paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) in the heart of ISO-administered rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt) and AA (80 mg/kg b.wt) orally for 6 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to individual drug treated groups. The combination of FA and AA preserved the membrane integrity by mitigating the oxidative stress and associated cellular damage more effectively when compared to individual treatment groups. In our study, the protection conferred by FA and AA might be through the nitric oxide pathway and by their ability of quenching free radicals. In conclusion, these findings indicate the synergistic modulation of lysosomal hydrolases and membrane phosphatases by the combination of FA and AA.

  20. Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini.

    PubMed

    McCartney, Andrew W; Dyer, John M; Dhanoa, Preetinder K; Kim, Peter K; Andrews, David W; McNew, James A; Mullen, Robert T

    2004-01-01

    Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER). To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where -Phi- are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.

  1. Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms.

    PubMed

    Harper, April D; Bar-Peled, Maor

    2002-12-01

    UDP-xylose (Xyl) is an important sugar donor for the synthesis of glycoproteins, polysaccharides, various metabolites, and oligosaccharides in animals, plants, fungi, and bacteria. UDP-Xyl also feedback inhibits upstream enzymes (UDP-glucose [Glc] dehydrogenase, UDP-Glc pyrophosphorylase, and UDP-GlcA decarboxylase) and is involved in its own synthesis and the synthesis of UDP-arabinose. In plants, biosynthesis of UDP-Xyl is catalyzed by different membrane-bound and soluble UDP-GlcA decarboxylase (UDP-GlcA-DC) isozymes, all of which convert UDP-GlcA to UDP-Xyl. Because synthesis of UDP-Xyl occurs both in the cytosol and in membranes, it is not known which source of UDP-Xyl the different Golgi-localized xylosyltransferases are utilizing. Here, we describe the identification of several distinct Arabidopsis genes (named AtUXS for UDP-Xyl synthase) that encode functional UDP-GlcA-DC isoforms. The Arabidopsis genome contains five UXS genes and their protein products can be subdivided into three isozyme classes (A-C), one soluble and two distinct putative membrane bound. AtUxs from each class, when expressed in Escherichia coli, generate active UDP-GlcA-DC that converts UDP-GlcA to UDP-Xyl. Members of this gene family have a large conserved C-terminal catalytic domain (approximately 300 amino acids long) and an N-terminal variable domain differing in sequence and size (30-120 amino acids long). Isoforms of class A and B appear to encode putative type II membrane proteins with their catalytic domains facing the lumen (like Golgi-glycosyltransferases) and their N-terminal variable domain facing the cytosol. Uxs class C is likely a cytosolic isoform. The characteristics of the plant Uxs support the hypothesis that unique UDP-GlcA-DCs with distinct subcellular localizations are required for specific xylosylation events.

  2. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    PubMed Central

    Herbert, Andrew S; Heffron, Lynn; Sundick, Roy; Roberts, Paul C

    2009-01-01

    Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4) fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective. PMID:19393093

  3. Reconstitution of Purified Acetylcholine Receptors with Functional Ion Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M.

    1980-05-01

    Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16± 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35± 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

  4. Channel opening motion of alpha7 nicotinic acetylcholine receptor as suggested by normal mode analysis.

    PubMed

    Cheng, Xiaolin; Lu, Benzhuo; Grant, Barry; Law, Richard J; McCammon, J Andrew

    2006-01-13

    The gating motion of the human nicotinic acetylcholine receptor (nAChR) alpha7 was investigated with normal mode analysis (NMA) of two homology models. The first model, referred to as model I, was built from both the Lymnaea stagnalis acetylcholine binding protein (AChBP) and the transmembrane (TM) domain of the Torpedo marmorata nAChR. The second model, referred to as model C, was based solely on the recent electron microscopy structure of the T. marmorata nAChR. Despite structural differences, both models exhibit nearly identical patterns of flexibility and correlated motions. In addition, both models show a similar global twisting motion that may represent channel gating. The similar results obtained for the two models indicate that NMA is most sensitive to the contact topology of the structure rather than its finer detail. The major difference between the low-frequency motions sampled for the two models is that a symmetrical pore-breathing motion, favoring channel opening, is present as the second most dominant motion in model I, whilst largely absent from model C. The absence of this mode in model C can be attributed to its less symmetrical architecture. Finally, as a further goal of the present study, an approximate open channel model, consistent with many experimental findings, has been produced.

  5. Investigation of Single-Stage Modified Turbine of Mark 25 Torpedo Power Plant

    NASA Technical Reports Server (NTRS)

    Hoyt, Jack W.

    1947-01-01

    Efficiency investigations have been made on a single-stage modification of the turbine of a Mark 25 aerial torpedo to determine the performance of the unit with five different turbine nozzles. The output of the turbine blades was computed by analyzing the windage and mechanical-friction losses of the unit. The turbine was faund to be most efficient with a cast nozzle having sharp-edged inlets to the nine nozzle ports. An analysis af the effectiveness af the first and second stages of the standard Mark 25 torpedo turbine indicates that the first- stage turbine contributes nearly all the brake power produced at blade-jet speed ratios above 0.26.

  6. Mechanical characterisation of the TorPeDO: a low frequency gravitational force sensor

    NASA Astrophysics Data System (ADS)

    McManus, D. J.; Forsyth, P. W. F.; Yap, M. J.; Ward, R. L.; Shaddock, D. A.; McClelland, D. E.; Slagmolen, B. J. J.

    2017-07-01

    Newtonian noise is likely to be a future challenge at low frequencies for Advanced LIGO and other second generation gravitational wave detectors. We present the TorPeDO system: a dual torsion pendulum sensor designed to measure local gravitational forces to high precision. Gravitational forces induce a differential rotation between the two torsion beams, which is measured with an optical read-out. Both torsion pendulums have a common suspension point, tunable centre of mass, and resonant frequency. This produces a high level of mechanical common mode noise cancellation. We report on a controls prototype of the TorPeDO system, presenting the frequency response and tuning range of both pendulums. A noise budget and mechanical cross-coupling model for this system are also presented. We demonstrate frequency tuning of the two torsion pendulums to a difference of 4.3 μHz.

  7. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    SciTech Connect

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  8. Torpedo electromotor system development: neuronal cell death and electric organ development in the fourth branchial arch.

    PubMed

    Fox, G Q; Richardson, G P; Kirk, C

    1985-06-08

    The fourth branchial arch of Torpedo marmorata has been examined at the light and electron microscopic level during development. Of interest was the determination of the extent of electric organ tissue reported to be present in this arch and its possible relationship to electromotoneuron cell death in the electric lobes. The main electric organ of the torpedo is derived from the hyoid and first three branchial arches and is innervated by four major electromotor nerves. Extensive electromotoneuron cell death occurs in the electric lobes and most notably in the posterior poles. This feature could be due to a tendency for these neurons to innervate the fourth branchial arch where little or no electric tissue is formed. Our findings support this conclusion but are not entirely consistent with the idea that a population mismatch has occurred. This is because cell death precedes the genesis of the target cells. The presence of innervated differentiated electric tissue in this arch is also reported, leading to the conclusion that Torpedo marmorata possesses an accessory electric organ.

  9. Trypanosoma evansi: pharmacological evidence of a nicotinic acetylcholine receptor.

    PubMed

    Portillo, R; Bruges, G; Delgado, D; Betancourt, M; Mijares, A

    2010-06-01

    The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor. After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a Kd1 value of 29.6+/-5.72 nM and a Kd2 value of 315.9+/-26.6 nM, which is similar to the Kd value reported for the alpha4 nicotinic receptor. The Hill coefficients were determined to be an n1 of 1.2+/-0.3 and an n2 of 4.2+/-1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  10. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

    SciTech Connect

    Lentz, T.L. )

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similar segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.

  11. The nicotinic acetylcholine receptor: Binding of nitroxide analogs of a local anesthetic and a photoactivatable analog of phosphatidylserine

    SciTech Connect

    Blanton, M.P.

    1989-01-01

    Electron spin resonance was used to contrast the accessibility of tertiary and quaternary amine local anesthetics to their high affinity binding site in the desensitized Torpedo californica acetylcholine receptor (AchR). Preincubation of AchR-rich membranes with agonist resulted in a substantial reduction in the initial association of the quaternary amine local anesthetic C6SLMEI with the receptor. The time-dependent reduction in association follows a biphasic exponential function having rate constants of 0.19 min{sup {minus}1} and 0.03 min{sup {minus}1}. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AchR. The results are modeled in two ways: (1) A charge gate near the channel mouth in the desensitized receptor limits access of the permanently charged cationic local anesthetic (C6SLMEI), but not for the uncharged form of the tertiary amine anesthetic C6SL. (2) A hydrophobic pathway, possibly through a corridor in the annular lipid surrounding receptor subunits, allows the uncharged form of C6SL to reach the high affinity binding site in the AchR. A photoactivatable analog of phosphatidylserine {sup 125}I 4-azido salicylic acid-phosphatidylserine ({sup 125}I ASA-PS) was use to label both Torpedo californica acetylcholine receptor-rich membranes and reconstituted AchR membranes. All four subunits of the AchR were found to incorporate label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporate {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. Eighty-one per cent of the incorporated label was localized to 11.7 and 10.1 kdal V8 cleavage fragments.

  12. Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation.

    PubMed

    Adachi, O; Fujii, Y; Ano, Y; Moonmangmee, D; Toyama, H; Shinagawa, E; Theeragool, G; Lotong, N; Matsushita, K

    2001-01-01

    To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be

  13. Increased soluble and membrane-bound PD-L1 contributes to immune regulation and disease progression in patients with tuberculous pleural effusion

    PubMed Central

    Pan, Xue; Zhong, Anyuan; Xing, Yufei; Shi, Minhua; Qian, Bin; Zhou, Tong; Chen, Yongjing; Zhang, Xueguang

    2016-01-01

    Soluble and membrane-bound programmed death ligand-1 (sPD-L1 and mPD-L1, respectively) have been demonstrated to participate in the immune suppression of non-small cell lung cancer. However, the contribution of sPD-L1 and mPD-L1 to immune regulation and disease progression in patients with pleural effusions remains unknown. The present study evaluated the levels of sPD-L1 and membrane-bound PD-1/PD-L1 in the peripheral blood and pleural effusions of patients with tuberculous pleural effusion (TPE), malignant pleural effusion (MPE) and non-tuberculous non-malignant pleural effusion (n-TB n-M). Furthermore, selected T lymphocytes and cluster of differentiation (CD)14+ monocytes were co-cultured to investigate the potential effect of the PD-1/PD-L1 pathway in TPE. Levels of sPD-L1 and PD-L1 on CD14+ monocytes were increased in the TPE group, as compared with the MPE and n-TB n-M groups. Furthermore, sPD-L1 levels and the expression levels of PD-L1 on CD14+ monocytes were demonstrated to be positively correlated with interferon (IFN)-γ concentration in pleural effusions. Therefore, IFN-γ may increase the expression of PD-L1 on CD14+ monocytes in vitro. Cell counting kit-8 analysis demonstrated that anti-PD-L1 antibody was able to partially reverse the proliferation of T lymphocytes in the co-culture system. The results of the present study indicated that sPD-L1 or mPD-L1 are associated with the immune regulation and disease progression of TPE, and may serve as possible biomarkers of TPE. Furthermore, sPD-L1 and the PD-1/PD-L1 pathway of TPE may be associated with the Th1 immune response; therefore, an anti-PD-1/PD-L1 pathway suggests a potential immune therapy strategy for the treatment of TPE. PMID:27698705

  14. Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

    PubMed

    Leong, Ron L; Xing, Hong; Braekman, Jean-Claude; Kem, William R

    2015-10-01

    Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more

  15. Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors.

    PubMed

    Vulfius, Catherine A; Spirova, Ekaterina N; Serebryakova, Marina V; Shelukhina, Irina V; Kudryavtsev, Denis S; Kryukova, Elena V; Starkov, Vladislav G; Kopylova, Nina V; Zhmak, Maxim N; Ivanov, Igor A; Kudryashova, Ksenia S; Andreeva, Tatyana V; Tsetlin, Victor I; Utkin, Yuri N

    2016-10-01

    Phospholipase A2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3-10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC50 of about 50 μM and 250 μM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC50 of about 3 μM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 μM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC50 of 20.6 ± 3.93 μM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 μM, the value being close to IC50 on α7 nAChR. These data are in

  16. Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene

    PubMed Central

    Fukaya, Masahiro; Tayama, Kenji; Tamaki, Toshimi; Tagami, Haruko; Okumura, Hajime; Kawamura, Yoshiya; Beppu, Teruhiko

    1989-01-01

    A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH2-terminal β-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively. Images PMID:16347820

  17. The Membrane-Bound Transcription Factor CREB3L1 Is Activated in Response to Virus Infection to Inhibit Proliferation of Virus-Infected Cells

    PubMed Central

    Denard, Bray; Seemann, Joachim; Chen, Qiuyue; Gay, Austin; Huang, Hua; Chen, Yan; Ye, Jin

    2011-01-01

    Summary CREB3L1/OASIS is a cellular transcription factor synthesized as a membrane-bound precursor and activated by regulated intramembrane proteolysis in response to stimuli like ER stress. Comparing gene expression between Huh7 subclones that are permissive for hepatitis C virus (HCV) replication versus the non-permissive parental Huh7 cells, we identified CREB3L1 as a cellular factor that inhibits proliferation of virus-infected cells. Upon infection with diverse DNA and RNA viruses including murine γ-herpesvirus 68, HCV, West Nile virus (WNV) and Sendai virus, CREB3L1 was proteolytically cleaved, allowing its NH2-terminus to enter the nucleus to induce multiple genes encoding inhibitors of the cell cycle to block cell proliferation. Consistent with this, we observed a necessity for CREB3L1 expression to be silenced in proliferating cells that harbor replicons of HCV or WNV. Our results indicate that CREB3L1 may play an important role in limiting virus spread by inhibiting proliferation of virus-infected cells. PMID:21767813

  18. Krypton Derivatization of an O2 -Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport.

    PubMed

    Kalms, Jacqueline; Schmidt, Andrea; Frielingsdorf, Stefan; van der Linden, Peter; von Stetten, David; Lenz, Oliver; Carpentier, Philippe; Scheerer, Patrick

    2016-04-25

    [NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

  19. Recognition of Membrane-Bound Fusion-Peptide/MPER Complexes by the HIV-1 Neutralizing 2F5 Antibody: Implications for Anti-2F5 Immunogenicity

    PubMed Central

    Huarte, Nerea; Araujo, Aitziber; Arranz, Rocio; Lorizate, Maier; Quendler, Heribert; Kunert, Renate; Valpuesta, José M.; Nieva, José L.

    2012-01-01

    The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen. PMID:23285173

  20. Purification and characterization of membrane-bound 3-dehydroshikimate dehydratase from Gluconobacter oxydans IFO 3244, a new enzyme catalyzing extracellular protocatechuate formation.

    PubMed

    Shinagawa, Emiko; Adachi, Osao; Ano, Yoshitaka; Yakushi, Toshiharu; Matsushita, Kazunobu

    2010-01-01

    3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6-8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.

  1. Newly discovered viral E3 ligase pK3 induces endoplasmic reticulum-associated degradation of class I major histocompatibility proteins and their membrane-bound chaperones.

    PubMed

    Herr, Roger A; Wang, Xiaoli; Loh, Joy; Virgin, Herbert W; Hansen, Ted H

    2012-04-27

    Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.

  2. Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION.

    PubMed

    Hutcheson, S W; Buchanan, B B

    1980-12-01

    The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H(2)O as electron donor for the photoreduction of NADP, (b) NADP replaced O(2) as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O(2) at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.The physiological mechanism for the activation of latent polyphenol oxidase remains an unanswered question. Present results suggest that activation could occur through either acidification or the release of free fatty acids.

  3. Purification of a Membrane-Bound UDP-Glucose:Sterol [beta]-D-Glucosyltransferase Based on Its Solubility in Diethyl Ether.

    PubMed Central

    Warnecke, D. C.; Heinz, E.

    1994-01-01

    Membrane-bound UDP-glucose:sterol [beta]-D-glucosyltransferase (UDPG-SGTase) catalyzes the formation of steryl glucosides from UDP-glucose and free sterols. This enzyme was purified from etiolated oat shoots (Avena sativa L. cv Alfred) in five steps. UDPG-SGTase was solubilized from a microsomal fraction with the detergent n-octyl-[beta]-D-thioglucopyranoside and then extracted into diethyl ether. Subsequent removal of the organic solvent, resolubilization with an aqueous buffer, and two column chromatographic steps on Q-Sepharose and Blue Sepharose resulted in a 12,500-fold overall purification. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation revealed a 56-kD protein band, the intensity of which correlated with enzyme activity in the respective fractions. Polyclonal antibodies raised against this 56-kD protein did not inhibit enzyme activity but specifically bound to the native UDPG-SGTase. These results suggest that the 56-kD protein represents the UDPG-SGTase. The purified enzyme was specific for UDP-glucose (Km = 34 [mu]M), for which UDP was a competitive inhibitor (inhibitor constant = 47 [mu]M). In contrast to the specificity with regard to the glycosyl donor, UDPG-SGTase utilized all tested sterol acceptors, such as [beta]-sitosterol, cholesterol, stigmasterol, and ergosterol. PMID:12232266

  4. Diverse in vivo effects of soluble and membrane-bound M-CSF on tumor-associated macrophages in lymphoma xenograft model.

    PubMed

    Liao, Jinfeng; Feng, Wenli; Wang, Rong; Ma, Shihui; Wang, Lina; Yang, Xiao; Yang, Feifei; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

    2016-01-12

    Macrophage colony-stimulating factor (M-CSF) is an important cytokine for monocyte/macrophage lineage. Secretory M-CSF (sM-CSF) and membrane-bound M-CSF (mM-CSF) are two major alternative splicing isoforms. The functional diversity of these isoforms in the activation of tumor-associated macrophages (TAMs), especially in lymphoma microenvironment, has not been documented. Here, we studied the effects of M-CSF isoforms on TAMs in xenograft mouse model. More infiltrating TAMs were detected in microenvironment with mM-CSF and sM-CSF. TAMs could be divided into three subpopulations based on their expression of CD206 and Ly6C. While sM-CSF had greater potential to recruit and induce differentiation of TAMs and TAM subpopulations, mM-CSF had greater potential to induce proliferation of TAMs and TAM subpopulations. Though both isoforms educated TAMs and TAM subpopulations to M2-like macrophages, mM-CSF and sM-CSF induced different spectrums of phenotype-associated genes in TAMs and TAM subpopulations. These results suggested the diverse effects of M-CSF isoforms on the activation of TAMs and TAM subpopulations in lymphoma microenvironments.

  5. Hydrogen evolution of Enterobacter aerogenes depending on culture pH: mechanism of hydrogen evolution from NADH by means of membrane-bound hydrogenase.

    PubMed

    Tanisho, S; Kamiya, N; Wakao, N

    1989-01-26

    The pH dependency of cell mass productivity, the hydrogen evolution rate and the yield of hydrogen from glucose was measured by controlling the pH of the culture automatically. The cell mass productivity of Enterobacter aerogenes increased in a linear fashion up to a pH value of approx. 7.0. In contrast, both the evolution rate and the yield of hydrogen showed convex relationships up to a pH value of 7.0, both having maximum values at a pH of approx. 5.8. The maximum evolution rate was approx. 11.3 mmol H2 per g dry cell per h at 38 degrees C. A hypothetical mechanism for hydrogen evolution was proposed by taking our results and other research work into consideration. The proposed mechanism of hydrogen evolution was that NADH was oxidized on the inside surface of the cell membrane and protons were reduced on the outside surface by means of membrane-bound hydrogenase. This mechanism explains in a thermodynamic context the relation between the activity of the hydrogen evolution and the pH of the culture.

  6. Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain.

    PubMed

    Liu, B; Hassler, D F; Smith, G K; Weaver, K; Hannun, Y A

    1998-12-18

    Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine- and stress-induced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The Km of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.

  7. Genome-wide identification of membrane-bound fatty acid desaturase genes in Gossypium hirsutum and their expressions during abiotic stress

    PubMed Central

    Feng, Jiyu; Dong, Yating; Liu, Wei; He, Qiuling; Daud, M. K.; Chen, Jinhong; Zhu, Shuijin

    2017-01-01

    Membrane-bound fatty acid desaturases (FADs) are of great importance and play multiple roles in plant growth and development. In the present study, 39 full-length FAD genes, based on database searches, were identified in tetraploid upland cotton (Gossypium hirsutum L.) and were phylogenetically clustered into four subfamilies. Genomic localization revealed that 34 genes were mapped on 22 chromosomes, and five genes were positioned on the scaffold sequences. The FAD genes of G. hirsutum in the same subfamily had similar gene structures. The structures of paralogous genes were considerably conserved in exons number and introns length. It was suggested that the FAD gene families in G. hirsutum might be duplicated mainly by segmental duplication. Moreover, the FAD genes were differentially expressed in different G. hirsutum tissues in response to different levels of salt and cold stresses, as determined by qRT-PCR analysis. The identification and functional analysis of FAD genes in G. hirsutum may provide more candidate genes for genetic modification. PMID:28374822

  8. Differences in the effect of phosphatidylinositol 4,5-bisphosphate on the hydrolytic and transphosphatidylation activities of membrane-bound phospholipase D from poppy seedlings.

    PubMed

    Oblozinsky, Marek; Bezakova, Lydia; Mansfeld, Johanna; Heilmann, Ingo; Ulbrich-Hofmann, Renate

    2013-08-01

    The hydrolytic activity of phospholipase D (PLD) yielding phosphatidic acid from phosphatidylcholine and other glycerophospholipids is known to be involved in many cellular processes. In contrast, it is not clear whether the competitive transphosphatidylation activity of PLD catalyzing the head group exchange of phospholipids has a natural function. In poppy seedlings (Papaver somniferum L.) where lipid metabolism and alkaloid synthesis are closely linked, five isoenzymes with different substrate and hydrolysis/transphosphatidylation selectivities have been detected hitherto. A membrane-bound PLD, found in microsomal fractions of poppy seedlings, is active at micromolar concentrations of Ca(2+) ions and needs phosphatidylinositol 4,5-bisphosphate (PIP2) as effector in the hydrolysis of phosphatidylcholine (PC). The optimum PIP2 concentration at 1.2 mol% of the concentration of the substrate PC indicates a specific activation effect. Transphosphatidylation with glycerol, ethanolamine, l-serine, or myo-inositol as acceptor alcohols is also activated by PIP2, however, with an optimum concentration at 0.6-0.9 mol%. In contrast to hydrolysis, a basic transphosphatidylation activity occurs even in the absence of PIP2, suggesting a different fine-tuning of the two competing reactions.

  9. Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

    PubMed

    Qian, Li; Ji, Ming-Chun; Pan, Xin-Yuan; Gong, Wei-Juan; Tian, Fang; Duan, Qiu-Fang

    2011-05-01

    Interleukin 15 (IL-15) is a pivotal cytokine for the proliferation and activation of a specific group of immune cells such as natural killer (NK), IFN-producing killer dendritic cells (IKDC) and CD8 T cells. RAE-1ε, the ligand for the activating NKG2D receptor, which also play an important role in the proliferation and activation of NK cells and IKDCs. In this study, a membrane-bound form of IL-15 (termed mb15) encoding sequence and RAE-1ε gene were obtained by SOE-PCR or PCR amplification. The amplified mb15 and RAE-1ε gene were then digested and inserted into the multiple cloning site1 (MCS1) and MCS2 of pVITRO2-mcs vector, respectively. A recombinant eukaryotic expression vector for co-expression of mb15 and RAE-1ε was successfully constructed. After it was transfected to BaF3 cells, the expression of IL-15 and RAE-1ε in recombinant BaF3/mb15/RAE-1ε cells were verified by RT-PCR, western blot and FCM analysis. Furthermore, BaF3/mb15/RAE-1ε cells had the ability of promoting NK cells proliferation and IFN-γ secretion. In conclusion, BaF3/mb15/RAE-1ε cells were successfully constructed, which is very useful for further studies, especially for the expansion and activation of certain subsets of immune cells such as NK cells and IKDCs.

  10. Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

  11. Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus.

    PubMed

    Raissi, Aram J; Staudenmaier, Emily K; David, Serena; Hu, Linda; Paradis, Suzanne

    2013-11-01

    While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process. © 2013.

  12. Detection and phylogenetic analysis of the membrane-bound nitrate reductase (Nar) in pure cultures and microbial communities from deep-sea hydrothermal vents.

    PubMed

    Pérez-Rodríguez, Ileana; Bohnert, Kenneth A; Cuebas, Mariola; Keddis, Ramaydalis; Vetriani, Costantino

    2013-11-01

    Over the past few years the relevance of nitrate respiration in microorganisms from deep-sea hydrothermal vents has become evident. In this study, we surveyed the membrane-bound nitrate reductase (Nar) encoding gene in three different deep-sea vent microbial communities from the East Pacific Rise and the Mid-Atlantic Ridge. Additionally, we tested pure cultures of vent strains for their ability to reduce nitrate and for the presence of the NarG-encoding gene in their genomes. By using the narG gene as a diagnostic marker for nitrate-reducing bacteria, we showed that nitrate reductases related to Gammaproteobacteria of the genus Marinobacter were numerically prevalent in the clone libraries derived from a black smoker and a diffuse flow vent. In contrast, NarG sequences retrieved from a community of filamentous bacteria located about 50 cm above a diffuse flow vent revealed the presence of a yet to be identified group of enzymes. 16S rRNA gene-inferred community compositions, in accordance with previous studies, showed a shift from Alpha- and Gammaproteobacteria to Epsilonproteobacteria as the vent fluids become warmer and more reducing. Based on these findings, we argue that Nar-catalyzed nitrate reduction is likely relevant in temperate and less reducing environments where Alpha- and Gammaproteobacteria are more abundant and where nitrate concentrations reflect that of background deep seawater.

  13. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

    PubMed Central

    Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

    2014-01-01

    This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111

  14. Retroviral gene transfer into primary human NK cells activated by IL-2 and K562 feeder cells expressing membrane-bound IL-21.

    PubMed

    Streltsova, Maria A; Barsov, Eugene; Erokhina, Sofia A; Kovalenko, Elena I

    2017-11-01

    Natural killer (NK) cells are capable of rapidly recognizing and efficiently killing tumor cells. This makes them a potentially promising agent for cancer immunotherapy. Additional genetic modifications of NK cells may further improve their anti-tumor efficacy. Numerous technical challenges associated with gene delivery into NK cells have significantly tempered this approach. We achieved efficient retroviral vector transduction of primary human NK cells that were stimulated by a combination of IL-2 and engineered K562 cells expressing membrane-bound IL-21. The activated NK cells were in less differentiated state and expressed NK cell activation receptors NKG2D, NKp30, CD16, and were highly HLA-DR-positive. This NK cell population was highly susceptible to the transduction by both GFP- and NGFR-expressing retroviral vectors, with transduction efficiency exceeding 50%. More mature CD57(+) NK cell population was generally resistant to retroviral vector transduction because of poor response to the stimulation. Our findings may facilitate retroviral vector-mediated genetic engineering of human primary NK cells for future immunotherapies. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Localization and function of the membrane-bound riboflavin in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae.

    PubMed

    Casutt, Marco S; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-08-27

    The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na(+) across the bacterial membrane. The Na(+)-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na(+)-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na(+)-NQR is discussed.

  16. The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD).

    PubMed

    Bateman, A; Bycroft, M

    2000-06-16

    The LysM domain is a widespread protein module. It was originally identified in enzymes that degrade bacterial cell walls but is also present in many other bacterial proteins. Several proteins that contain the domain, such as Staphylococcal IgG binding proteins and Escherichia coli intimin, are involved in bacterial pathogenesis. LysM domains are also found in some eukaryotic proteins, apparently as a result of horizontal gene transfer from bacteria. The available evidence suggests that the LysM domain is a general peptidoglycan-binding module. We have determined the structure of this domain from E. coli membrane-bound lytic murein transglycosylase D. The LysM domain has a betaalphaalphabeta secondary structure with the two helices packing onto the same side of an anti- parallel beta sheet. The structure shows no similarity to other bacterial cell surface domains. A potential binding site in a shallow groove on surface of the protein has been identified. Copyright 2000 Academic Press.

  17. An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria.

    PubMed

    Wang, Jian-Gong; Xia, Fei; Zeleke, Jemaneh; Zou, Bin; Rhee, Sung-Keun; Quan, Zhe-Xue

    2017-03-01

    The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. ANKYRIN REPEAT-CONTAINING PROTEIN 2A is an essential molecular chaperone for peroxisomal membrane-bound ASCORBATE PEROXIDASE3 in Arabidopsis.

    PubMed

    Shen, Guoxin; Kuppu, Sundaram; Venkataramani, Sujatha; Wang, Jing; Yan, Juqiang; Qiu, Xiaoyun; Zhang, Hong

    2010-03-01

    Arabidopsis thaliana ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) interacts with peroxisomal membrane-bound ASCORBATE PEROXIDASE3 (APX3). This interaction involves the C-terminal sequence of APX3 (i.e., a transmembrane domain plus a few basic amino acid residues). The specificity of the AKR2A-APX3 interaction suggests that AKR2A may function as a molecular chaperone for APX3 because binding of AKR2A to the transmembrane domain can prevent APX3 from forming aggregates after translation. Analysis of three akr2a mutants indicates that these mutant plants have reduced steady state levels of APX3. Reduced expression of AKR2A using RNA interference also leads to reduced steady state levels of APX3 and reduced targeting of APX3 to peroxisomes in plant cells. Since AKR2A also binds specifically to the chloroplast OUTER ENVELOPE PROTEIN7 (OEP7) and is required for the biogenesis of OEP7, AKR2A may serve as a molecular chaperone for OEP7 as well. The pleiotropic phenotype of akr2a mutants indicates that AKR2A plays many important roles in plant cellular metabolism and is essential for plant growth and development.

  19. Engraftment of human HSCs in nonirradiated newborn NOD-scid IL2rγnull mice is enhanced by transgenic expression of membrane-bound human SCF

    PubMed Central

    Racki, Waldemar J.; Leif, Jean; Burzenski, Lisa; Hosur, Vishnu; Wetmore, Amber; Gott, Bruce; Herlihy, Mary; Ignotz, Ronald; Dunn, Raymond; Shultz, Leonard D.; Greiner, Dale L.

    2012-01-01

    Immunodeficient mice engrafted with human HSCs support multidisciplinary translational experimentation, including the study of human hematopoiesis. Heightened levels of human HSC engraftment are observed in immunodeficient mice expressing mutations in the IL2-receptor common γ chain (IL2rg) gene, including NOD-scid IL2rγnull (NSG) mice. Engraftment of human HSC requires preconditioning of immunodeficient recipients, usually with irradiation. Such preconditioning increases the expression of stem cell factor (SCF), which is critical for HSC engraftment, proliferation, and survival. We hypothesized that transgenic expression of human membrane-bound stem cell factor Tg(hu-mSCF)] would increase levels of human HSC engraftment in nonirradiated NSG mice and eliminate complications associated with irradiation. Surprisingly, detectable levels of human CD45+ cell chimerism were observed after transplantation of cord blood–derived human HSCs into nonirradiated adult as well as newborn NSG mice. However, transgenic expression of human mSCF enabled heightened levels of human hematopoietic cell chimerism in the absence of irradiation. Moreover, nonirradiated NSG-Tg(hu-mSCF) mice engrafted as newborns with human HSCs rejected human skin grafts from a histoincompatible donor, indicating the development of a functional human immune system. These data provide a new immunodeficient mouse model that does not require irradiation preconditioning for human HSC engraftment and immune system development. PMID:22246028

  20. Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

    PubMed

    Nguyen, Nga T; Yatabe, Takeshi; Yoon, Ki-Seok; Ogo, Seiji

    2014-10-01

    Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

  1. Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

    SciTech Connect

    Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

    1985-08-01

    The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

  2. Isolation and structural characterization of two water-borne pheromones from Euplotes crassus, a ciliate commonly known to carry membrane-bound pheromones.

    PubMed

    Alimenti, Claudio; Vallesi, Adriana; Federici, Sergio; di Giuseppe, Graziano; Fernando, Dini; Carratore, Vitale; Luporini, Pierangelo

    2011-01-01

    Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome. © 2011 The Author(s). Journal of Eukaryotic Microbiology© 2011 International Society of Protistologists.

  3. Difference in NaCl tolerance of membrane-bound 5'-nucleotidases purified from deep-sea and brackish water Shewanella species.

    PubMed

    Kuribayashi, Taka-Aki; Fujii, Sotaro; Masanari, Misa; Yamanaka, Masaru; Wakai, Satoshi; Sambongi, Yoshihiro

    2017-01-03

    Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5'-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl2 for their activities, and they exhibited the same pH dependency and substrate specificity for 5'-nucleotides. However, S. violacea 5'-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5'-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5'-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.

  4. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    SciTech Connect

    Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi; Turk, Tom; Sepčić, Kristina; Benoit, Evelyne; Frangež, Robert

    2012-12-01

    APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.

  5. Tetanus toxin mechanism of action in Torpedo electromotor system: a study on different steps in the intoxication process.

    PubMed

    Herreros, J; Blasi, J; Arribas, M; Marsal, J

    1995-03-01

    The mechanism of action of tetanus toxin was characterized in the electromotor system of Torpedo marmorata either at peripheral and central nervous system. The consecutive steps of the intoxication pathway were observed: (i) [125I]tetanus toxin specifically bound to neuronal plasma membranes isolated both from electric organ and electric lobe of Torpedo, exhibiting one and two binding sites respectively; (ii) [125I]tetanus toxin was internalized into nerve terminals and retrogradely transported to the electric lobe after its injection in the electric organ; (iii) finally, intracellular effect of tetanus toxin was studied either at electric organ and electric lobe membrane fractions. In both preparations tetanus toxin cleaved synaptobrevin, as detected by immunoblotting methods. In conclusion, our findings exhibit the presence of two different populations of acceptors for tetanus toxin in central and peripheral nervous system and show that synaptobrevin cleavage may account for intracellular toxicity in Torpedo.

  6. NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1*

    PubMed Central

    Lyukmanova, Ekaterina N.; Shenkarev, Zakhar O.; Shulepko, Mikhail A.; Mineev, Konstantin S.; D'Hoedt, Dieter; Kasheverov, Igor E.; Filkin, Sergey Yu.; Krivolapova, Alexandra P.; Janickova, Helena; Dolezal, Vladimir; Dolgikh, Dmitry A.; Arseniev, Alexander S.; Bertrand, Daniel; Tsetlin, Victor I.; Kirpichnikov, Mikhail P.

    2011-01-01

    Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5–30 μm, ws-LYNX1 competed with 125I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μm ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μm caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding. PMID:21252236

  7. NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

    PubMed

    Lyukmanova, Ekaterina N; Shenkarev, Zakhar O; Shulepko, Mikhail A; Mineev, Konstantin S; D'Hoedt, Dieter; Kasheverov, Igor E; Filkin, Sergey Yu; Krivolapova, Alexandra P; Janickova, Helena; Dolezal, Vladimir; Dolgikh, Dmitry A; Arseniev, Alexander S; Bertrand, Daniel; Tsetlin, Victor I; Kirpichnikov, Mikhail P

    2011-03-25

    Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 μM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

  8. The influence of taipoxin on cholinergic synapses in the electric organ of Torpedo marmorata.

    PubMed

    Naef, W; Waser, P G

    1978-01-01

    The influences of the snake venom taipoxin on the cholinergic synapses of the electric organ of Torpedo mamorata were investigated. 1. In ultrathin sections presynaptic membrane indentations are noted under the influence of taipoxin/NaCl. 2. The presynaptic membranes often show small interruptions. 3. In taipoxin/horseradish peroxidase incubated organ pieces typical omega-shaped indentations are found, but on the postsynaptic side. 4. The vesicle density decreased under the influence of the taipoxin in contrast untreated cholinergic synapses. 5. In freeze etched preparations no membrane indentations and interruptions are observed, but only a small vesicle density was seen.

  9. Effect of First-Stage Blade Design on Performance of Mark 25 Torpedo Power Plant

    NASA Technical Reports Server (NTRS)

    Schum, Harold J.; Hoyt, Jack W.

    1948-01-01

    The effect of rotor-blade length, inlet angle, and shrouding was investigated with four different nozzles in a single-stage modification of the Mark 25 aerial-torpedo power plant. The results obtained with the five special rotor configurations are compared with those of the standard first-stage rotor with each nozzle. Each nozzle-rotor combination was operated at nominal pressure ratios of 8, 15 (design), and 20 over a range of speeds from 6000 rpm to the design speed of 18,000 rpm. Inlet temperature and pressure conditions of 1OOOo F and 95 pounds per square inch gage, respectively, were maintained constant for all runs.

  10. Investigation of Turbine of Mark 25 Torpedo Power Plant with Five Nozzle Designs

    NASA Technical Reports Server (NTRS)

    Hoyt, Jack W.; Kottas, Harry

    1947-01-01

    Efficiency investigations were made on the two-stage turbine from a Mark 25 aerial torpedo to determine the performance of the unit with five different turbine nozzles. The output of the turbine blades was computed by analyzing the windage and mechanical-friction losses of the unit. A method was developed for measuring the change in turbine clearances with changed operating conditions. The turbine was found to be most efficient with a cast nozzle having a sharp-edged inlet to the nine nozzle ports.

  11. Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

    PubMed

    Pica, A; Scacco, S; Papa, F; De Nitto, E; Papa, S

    2001-02-01

    A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

  12. Electrophysiological aspects of synaptic transmission at the electromotor junction of Torpedo marmorata.

    PubMed

    Erdelyi, L; Krenz, W D

    1984-01-01

    Miniature and stimulus-evoked electroplaque potentials were recorded in Torpedo electrocytes intracellularly and extracellularly and analysed quantitatively. Tetrodotoxin reversibly blocked stimulus evoked potentials but hardly affected spontaneous miniature potentials in amplitude and frequency. The quantum content of stimulus-evoked potentials varied between 150 and 400 in normal saline and decreased in low Ca2+ high Mg2+ solution. Quantal release conformed to binomial statistics and allowed determination of the release parameters p and n. Analysis of the time constant of decay of spontaneous miniature electroplaque currents showed variation around a mean of 0.75 +/- 0.16 msec (SD) which was greatly prolonged by application of neostigmine.

  13. The action of aminopyridine on the electromotor synapse of Torpedo marmorata.

    PubMed

    Erdélyi, L

    1983-09-09

    An investigation of the action of 4-aminopyridine (4-AP) on Torpedo electromotor synapses was performed using the microelectrode technique. 4-AP in concentrations of 1--10 micrometers increased the peak amplitude (p) and half-decay time (t 1/2) of the intracellularly recorded excitatory postsynaptic potentials (EEP), independent of alterations in miniature EPPs or membrane potential. 4-AP also increased the amplitude and time constant of decay (tau) of the excitatory postsynaptic current (EPC). Analysis of the results supports potentiated transmitter release and accumulation of the transmitter in the synaptic cleft as the mechanism by which 4-AP produces its effect.

  14. Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching.

    PubMed

    Takemura, Tsukasa; Hino, Satoshi; Okada, Mituru; Murata, Yuka; Yanagida, Hidehiko; Ikeda, Masaru; Yoshioka, Kazuo; Harris, Raymond C

    2002-06-01

    Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching. The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis. Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media. Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoelectron microscopy revealed that the initial recovery of cellular proHB-EGF was localized predominantly to areas of cell-cell attachment. No tubule-like structures were observed in similarly treated NRK

  15. [Dynamic changes of aciduric virulence factor membrane-bound proton-translocating ATPase of Streptococcus mutans in the development of dental caries].

    PubMed

    Gao, Jing; Huang, Wenming

    2016-04-01

    To observe the dynamic changes of membrane-bound proton-translocating ATPase (F-ATPase) in the development of dental caries, the expression of Streptococcus mutans F-ATPase under different pH concentrations and during the development of dental caries is analyzed. Streptococcus mutans cultured under different pH (pH4.0-7.0) concentrations and containing 5% glucose and no glucose containing BHI were collected. RNA was extracted. Subsequently, F-ATPase gene was detected using real-time polymerase chain reaction. Male Wistar rats were divided randomly into caries group and control group. The rats in the caries group were fed caries feed and 5% glucose water, whereas those of control group were fed normal feed. Total RNA was extracted from plaque samples, which were collected from rats' oral cavity every two weeks. F-ATPase gene was detected by real-time PCR. In the 11th week, the upper and lower jaw bone specimens of rats were taken, and molar caries damage assessed. The expression of F-ATPase in the caries group was higher than that in the control group (P<0.05). In addition, the gene was expressed highest in pH5.0 and the lowest in pH4.0 (P<0.05). 2) The expression of F-ATPase progressively increased during the caries development in both groups; expression in the caries group was higher than that in control group (P<0.05). Acid-resisting viru-lence factor F-ATPase is related closely with the incidence and development of dental caries.

  16. Molecular dynamics investigations of membrane-bound CYP2C19 polymorphisms reveal distinct mechanisms for peripheral variants by long-range effects on the enzymatic activity.

    PubMed

    Cui, Ying-Lu; Wu, Rong-Ling

    2017-06-01

    Increasing sophistication in methods used to account for human polymorphisms in susceptibility to drug metabolism has been one of the success stories in the prevention of adverse drug reactions. Genetic polymorphisms in drug-metabolizing enzymes can affect enzyme activity and cause differences in treatment response or drug toxicity. CYP2C19 is one of the most highly polymorphic CYP enzymes and acts on 10-15% of drugs in current clinical use. Despite the number of experimental analyses carried out for this system, the detailed structural basis for altered catalytic properties of polymorphic CYP2C19 variants remains unexplained at the atomic level. To this end, we have investigated the mutation effects on structural characteristics and tunnel geometry upon single point mutations to elucidate the underlying molecular mechanism for the enzymatic activity deficiencies by using the fully atomistic molecular dynamics (MD) simulations in their native, membrane-bound cellular environment. The obtained results demonstrate how significant sequence divergence causes heterogeneous variations, and further affects the shape and chemical properties of the substrate binding site. Principal component analysis (PCA) results combined with free energy calculations have revealed distinct mechanisms for different peripheral variants, implying a more complicated process for the decrease/loss of enzymatic activity upon the introduction of point mutations in CYP2C19 rather than simply structural changes of the region where the mutation is located. Overall, our present study provides important insights into the current pharmacogenetic knowledge of human drug-metabolizing CYP2C19 to understand the large inter-individual variability in drug clearance. The knowledge of heterogeneous variations in structural features could guide future experimental and computational work on efficient and safe drug treatment with better pharmacokinetic properties based on the common variant alleles of CYP genes

  17. Dimethoate induces kidney dysfunction, disrupts membrane-bound ATPases and confers cytotoxicity through DNA damage. Protective effects of vitamin E and selenium.

    PubMed

    Ben Amara, Ibtissem; Karray, Aida; Hakim, Ahmed; Ben Ali, Yassine; Troudi, Afef; Soudani, Nejla; Boudawara, Tahia; Zeghal, Khaled Mounir; Zeghal, Najiba

    2013-12-01

    Dimethoate (DM) is an organophosphate insecticide widely used in agriculture and industry and has toxic effects on non-target organisms especially mammalian. However, we still know little about DM-induced kidney injury and its alleviation by natural antioxidants. In the present study, selenium (Se), vitamin E, DM, Se+DM, vitamin E+DM, Se+vitamin E+DM were given to adult rats for 4 weeks. Plasma creatinine and uric acid, kidney MDA, PC, H2O2 and AOPP levels were higher, while Na(+)-K(+)-ATPase and LDH values were lower in the DM group than those of controls. A smear without ladder formation on agarose gel was shown in the DM group, indicating random DNA degradation and DM-induced genotoxicity. A decrease in kidney GSH, NPSH and plasma urea levels and an increase in GPx, SOD and catalase activities were observed in the DM group when compared to those of controls. Plasma cystatin C levels increased, indicating a decrease in glomerular filtration rate. When Se or vitamin E was added through diet, the biochemical parameters cited above were partially restored in Se+DM and vitamin E+DM than DM group. The joint effect of Se and vitamin E was more powerful against DM-induced oxidative stress and kidney dysfunction. The changes in biochemical parameters were substantiated by histological data. In conclusion, our results indicated a possible mechanism of DM-induced nephrotoxicity, where renal genotoxicity was noted, membrane-bound ATPases and plasma biomarkers were disturbed. Se and vitamin E ameliorated the toxic effects of this pesticide in renal tissue suggesting their role as potential antioxidants.

  18. Evidence for membrane-bound carbonic anhydrase in the air bladder of bowfin (Amia calva), a primitive air-breathing fish.

    PubMed

    Gervais, M R; Tufts, B L

    1998-07-01

    The purpose of this study was to examine the subcellular distribution and isoenzyme characteristics of carbonic anhydrase from the gills and respiratory air bladder of bowfin Amia calva, a primitive air-breathing fish. Separation of subcellular fractions by differential centrifugation revealed that the vast majority of carbonic anhydrase from the gills of bowfin originated from the cytoplasmic fraction. Washing of the gill microsomal pellet also indicated that the carbonic anhydrase originally associated with this pellet was largely due to contamination from the cytoplasmic fraction. Experiments with a carbonic anhydrase inhibitor, sulphanilamide, and the plasma carbonic anhydrase inhibitor from this species confirmed that the bowfin gill probably contains only one carbonic anhydrase isoenzyme which had properties resembling those of CA II. In contrast to the situation in the gills, a relatively large percentage (27%) of the total air bladder carbonic anhydrase was associated with the microsomal fraction. Washing of the air bladder microsomal pellet removed little of the carbonic anhydrase activity, indicating that most of the carbonic anhydrase in the microsomal fraction was associated with the membranes. Like the mammalian pulmonary CA IV isoenzyme, microsomal carbonic anhydrase from the bowfin air bladder was less sensitive to the bowfin plasma carbonic anhydrase inhibitor, sodium dodecylsulphate (SDS) and sulphanilamide than was cytoplasmic carbonic anhydrase from the air bladder. Microsomal carbonic anhydrase from the bowfin air bladder also resembled CA IV in that it appears to be anchored to the membrane via a phosphatidylinositol-glycan linkage which could be cleaved by phosphatidylinositol-specific phospholipase C. Taken together, these results suggest that a membrane-bound carbonic anhydrase isoenzyme resembling mammalian CA IV in terms of inhibition characteristics and membrane attachment is present in the air-breathing organ of one of the most primitive

  19. A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway1[OPEN

    PubMed Central

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J.

    2015-01-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  20. Properties, intracellular localization, and stage-specific expression of membrane-bound beta-glucosidase, BglM1, from Physarum polycephalum.

    PubMed

    Hayase, Masato; Maekawa, Akinori; Yubisui, Toshitsugu; Minami, Yoshiko

    2008-01-01

    Physarum polycephalum expresses a membrane-bound beta-glucosidase (BglM1) with a molecular mass of 130 kDa. The primary structure of BglM1 consists of a glycosyl hydrolase family 3 domain at an amino-terminal domain and a carboxyl-terminal region without homology to the sequence of known glycosidases. The latter region contains two calx-beta motifs known as Ca(2+)-binding sites; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. The molecular mass calculated from the amino acid sequence is 130 kDa, but that in the crude extract was estimated by SDS-PAGE to be 230 kDa, and decreased to 130 kDa during purification. However, when BglM1 was purified in the presence of calcium ion, the molecular mass remained 230 kDa. The biochemical characteristics of the 130- and 230-kDa BglM1 forms were analyzed: differences were found in the kinetic data for some substrates specific for both these enzymes; however, no difference was found in their intrinsic characteristics such as optimum pH and temperature. In addition, the molecular mass of native BglM1 with a calcium ion was estimated to be 1,000 kDa or larger by gel filtration. These results suggest that the calcium ion influences the conformation of BglM1. The evidence that BglM1 localizes on the plasma membrane of plasmodia was confirmed using immunofluorescence microscopy. Although Physarum BglM1 was expressed in microplasmodia and plasmodia, little expression was detected in other stages. BglM1 may have some function only in multinuclear cells.

  1. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    SciTech Connect

    Bansal, Sunil; Durrett, Timothy P.

    2016-11-08

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.

  2. Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium

    PubMed Central

    Matsumoto, H; Murakami, Y; Kataoka, K; Notomi, S; Mantopoulos, D; Trichonas, G; Miller, J W; Gregory, M S; Ksander, B R; Marshak-Rothstein, A; Vavvas, D G

    2015-01-01

    Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL−/−), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL−/− mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment. PMID:26583327

  3. Regulatory role of membrane-bound form interleukin-15 on human uterine microvascular endothelial cells in circulating CD16(-) natural killer cell extravasation into human endometrium.

    PubMed

    Kitaya, Kotaro; Yasuo, Tadahiro

    2013-09-01

    Interleukin (IL)-15 plays a major role in accumulation of unique CD16(-) natural killer (NK) cells in the human endometrium, partly via selective extravasation of peripheral blood (PB) counterparts from local microvascular circulation. While IL-15 exhibits a chemotactic activity for PB CD16(-) NK cells, IL-15 attenuates their binding capacity to dermatan sulfate, the major CD62L ligand expressed on human uterine microvascular endothelial cells (HUtMVECs). These findings suggest that premature action of IL-15 interferes with CD62L-dependent tethering/rolling of PB CD16(-) NK cells on HUtMVECs, which is an early critical process of leukocyte extravasation. In this study, we investigated the mechanisms underlying the IL-15 regulation in the initial CD62L-dependent contact between PB CD16(-) NK cells and HUtMVECs. Unlike other candidate molecules, recombinant IL-15 downregulated CD62L expression on freshly isolated PB CD16(-) NK cells. IL-12 and IL-10, the two known upregulators of CD62L on CD16(-) NK cells, were not detectable in HUtMVECs and endometrial perivascular stromal cells. Binding to immobilized dermatan sulfate increased surface IL-15 receptor-alpha chain expression on CD16(-) NK cells. Under ovarian steroid stimulation, IL-15 was detectable on the surface, but not in the supernatant, of cultured HUtMVECs. Ovarian steroid-induced IL-15 expression on HUtMVECs was not attenuated by chondroitinase ABC (which degrades chondroitin sulfate-A and -C and dermatan sulfate) or sodium acetate buffer (which dissociates cytokines from their cognate receptors). These results suggest that HUtMVECs secrete a less soluble form of IL-15 into local microcirculation. Instead, HUtMVECs bear a membrane-bound form IL-15 under the influence of ovarian steroids, which may be favorable for preventing downregulation of CD62L on PB CD16(-) NK cells and facilitating their initial contact with HUtMVECs.

  4. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: Resolution of the activity into soluble and membrane-bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-07-01

    The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelatase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzymen was sensitive to the sulfhydryl reagent N-ethylmaleimide (I{sub 50}, 20 {mu}M). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.

  5. Effect of 2-Hydroxy-4-methoxy Benzoic Acid Isolated from Hemidesmus indicus on Erythrocyte Membrane Bound Enzymes and Antioxidant Status in Streptozotocin-induced Diabetic Rats.

    PubMed

    Gayathri, M; Kannabiran, K

    2012-09-01

    In the present study, the effect of 2-hydroxy-4-methoxy benzoic acid isolated from roots of Hemisdesmus indicus on the erythrocyte membrane bound enzymes and antioxidant status in streptozotocin-induced diabetic rats was investigated. The streptozotocin-induced diabetic rats were treated with 2-hydroxy-4-methoxy benzoic acid (500 μg/kg/day) for 7 weeks by oral intubation and compared with glibenclamide, a standard hypoglycemic agent (100 mg/kg). The erythrocyte membrane was isolated and the activity of Na(+)/K(+)-dependent ATPases, Ca(2+)-ATPases, Mg(2+)-ATPases were determined. Superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, vitamins C, vitamin E, plasma reduced glutathione and erythrocyte glutathione, reduced glutathione content in the tissues was also assayed. Administration of 2-hydroxy-4-methoxy benzoic acid to diabetic rats significantly (F>0.05 and P<0.001) elevated the activity of total ATPases, Na(+)/k(+) ATPase, Mg(2+) ATPase and Ca(2+) ATPase to near normal level. The activities of catalase, superoxide dismutase and glutathione peroxidase and glutathione-S-transferase in erythrocytes were decreased significantly (F>0.05; P<0.001) in diabetic rats. Diabetic rats treated with 2-hydroxy-4-methoxy benzoic acid showed a significant (F>0.05; <0.001) increase in the enzymic antioxidants in erythrocytes. The elevated levels of vitamin E and low level of vitamin C and glutathione level in plasma and erythrocytes were observed in diabetic rats when compared to control rats and were restored significantly (F>0.05; P<0.001) after the administration of 2-hydroxy-4-methoxy benzoic acid. This study concludes administration of 2-hydroxy-4-methoxy benzoic acid supports the restoration of antioxidant defence, reduces the free radial production, lipid peroxidation and the glycosylation of haemoglobin in diabetic rats.

  6. Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein.

    PubMed

    DeMarco, Mari L; Daggett, Valerie

    2009-04-01

    The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo, there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrP(C)) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrP(C) as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrP(C), but they do appreciably modify the accessibility of the polypeptide surface PrP(C). In addition, the simulations of membrane-bound PrP(C) revealed likely recognition domains for the disease-initiating PrP(C):PrP(Sc) (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species.

  7. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  8. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells

    PubMed Central

    Prasad, Ram; Kappes, John C.; Katiyar, Santosh K.

    2016-01-01

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22phox and p47phox) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47phox protein and decreased levels of the membrane-bound p22phox protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues. PMID:26760964

  9. Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells.

    PubMed

    Prasad, Ram; Kappes, John C; Katiyar, Santosh K

    2016-02-16

    Overexpression of NADPH oxidase 1 (Nox1) in melanoma cells is often associated with increased migration/metastasis rate. To develop effective treatment options, we have examined the effect of honokiol, a phytochemical from Magnolia plant, on the migratory potential of human melanoma cell lines (A375, Hs294t, SK-Mel119 and SK-Mel28) and assessed whether Nox1 is the target. Using an in vitro cell migration assay, we observed that treatment of different melanoma cell lines with honokiol for 24 h resulted in a dose-dependent inhibition of cell migration that was associated with reduction in Nox1 expression and reduced levels of oxidative stress. Treatment of cells with N-acetyl-L-cysteine, an anti-oxidant, also inhibited the migration of melanoma cells. Treatment of cells with diphenyleneiodonium chloride, an inhibitor of Nox1, significantly decreased the migration ability of Hs294t and SK-Mel28 cells. Further, we examined the effect of honokiol on the levels of core proteins (p22(phox) and p47(phox)) of the NADPH oxidase complex. Treatment of Hs294t and SK-Mel28 cells with honokiol resulted in accumulation of the cytosolic p47(phox) protein and decreased levels of the membrane-bound p22(phox) protein, thus blocking their interaction and inhibiting Nox1 activation. Our in vivo bioluminescence imaging data indicate that oral administration of honokiol inhibited the migration/extravasation and growth of intravenously injected melanoma cells in internal body organs, such as liver, lung and kidney in nude mice, and that this was associated with an inhibitory effect on Nox1 activity in these internal organs/tissues.

  10. LPT1 encodes a membrane-bound O-acyltransferase involved in the acylation of lysophospholipids in the yeast Saccharomyces cerevisiae.

    PubMed

    Tamaki, Hisanori; Shimada, Atsushi; Ito, Yoshihiro; Ohya, Mihoko; Takase, Juri; Miyashita, Masahiro; Miyagawa, Hisashi; Nozaki, Hiroyuki; Nakayama, Reiko; Kumagai, Hidehiko

    2007-11-23

    Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

  11. Formation of membrane-bound inclusions and their associations with cytoplasmic channels in early prophase male meiocytes of Althaea rosea (L.) Cavan.

    PubMed

    Luo, Xin Juan; Liu, Xu Hao; Wang, Chong Ying; Wang, Xin Yu

    2008-04-01

    To characterize the cytoplasmic structure reorganization during plant meiosis, the male meiocytes of Althaea rosea (L.) Cavan were examined under the combination of light and electron microscopy. Light microscopic observation of the toluidine blue-stained thick resin sections of young anthers revealed that the meiocytes of sporogenous cell stage were extremely voluminous and variable in shape and division plane. The cell walls (CWs) between some meiocytes were discontinuous at one or several site(s). These discontinuous portions varied between 0.2 and 3.0 microm in length. In addition, it was found that some meiocytes were able to produce protuberances that extended into another meiocyte. When transversally sectioned, the protuberance extending to another cell looked like a small cell lying in another cell. Transmission electron microscopy (TEM) showed that there were many long flat ER cisternae that were actively wrapping around a portion of cytoplasm in the male meiocytes at the sporogenous cell stage. During pre-meiosis interphase and early prophase I, a number of huge (0.5-1.0 microm diameter) spherical membrane-bound inclusions (MBIs) lined by single or double layer(s) of membrane were formed, each membrane actually representing one tightly appressed endoplasmic reticulum (ER) cisterna. The MBIs contained many granular, lamellar and fibrillar structures, and even small MBIs. Moreover, it was found that the MBIs could associate with the cytoplasmic channels (CCs) on CWs to release their contents into the cytoplasm of the opposite cell or directly extend from one cell to another through the CC. Taking all the data together, it is suggested that association of the MBIs and other organelles with CCs possibly functions in eliminating the non-identity of cytoplasm of the male meiocytes caused probably by the random asymmetric division observed at sporogenous cell phase, so as to ensure production of a large number of identical functional male gametes required for

  12. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases[S

    PubMed Central

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  13. Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations

    PubMed Central

    1981-01-01

    Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed. PMID:6116718

  14. The Membrane Bound LRR Lipoprotein Slr, and the Cell Wall-Anchored M1 Protein from Streptococcus pyogenes Both Interact with Type I Collagen

    PubMed Central

    Bober, Marta; Mörgelin, Matthias; Olin, Anders I.; von Pawel-Rammingen, Ulrich; Collin, Mattias

    2011-01-01

    Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been indentified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20–30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors. PMID:21655249

  15. Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

    PubMed Central

    Cash, Derek J.; Hess, George P.

    1980-01-01

    Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine

  16. Expression of Membrane-Bound Human AminopeptidaseP as a Soluble Enzyme and an Investigation into Its Efficacy Towards Offering Protection Against the Toxicity of Chemical Warfare Nerve Agents

    DTIC Science & Technology

    2016-09-01

    histidine tag was also introduced in frame immediately before the stop codon. The PCR product was initially cloned into the expression plasmid pCMV 6...114-6. 13. Sprinkle, T.J., C. Caldwell, and J.W. Ryan, Cloning , chromosomal sublocalization of the human soluble aminopeptidase P gene (XPNPEP1...14. Venema, R.C., et al., Cloning and tissue distribution of human membrane-bound aminopeptidase P. Biochim Biophys Acta, 1997. 1354(1): p. 45-8

  17. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  18. Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor.

    PubMed

    Bracci, L; Antoni, G; Cusi, M G; Lozzi, L; Niccolai, N; Petreni, S; Rustici, M; Santucci, A; Soldani, P; Valensin, P E

    1988-09-01

    It has been reported that binding to muscle nicotinic acetylcholine receptor at the post-synaptic membrane is an important event of the rabies virus neurotropism. The binding site can be located within the 190-203 region of the virus glycoprotein sharing a high degree of homology with the "toxic loop" of the curare-mimetic snake neurotoxins. We have synthesized a tetradecapeptide corresponding to this glycoprotein region and used it, following conjugation with an immunogenic carrier to raise MAbs. We found that some MAbs raised against the peptide were able to recognize both the virus glycoprotein and the snake neurotoxin alpha-bungarotoxin; moreover, they can inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor extracted from the electric organs of Torpedo marmorata. On the basis of this cross-reactivity, we suggest that rabies virus glycoprotein and curare-mimetic snake neurotoxins share three-dimensionally similar structures in order to bind to the nicotinic cholinergic receptor. The potential use of the immunogenic properties of the peptide for the rational design of a synthetic vaccine against rabies is proposed.

  19. TORPEDO: Networked Access to Full-Text and Page-Image Representations of Physics Journals and Technical Reports.

    ERIC Educational Resources Information Center

    Atkinson, Roderick D.; Stackpole, Laurie E.

    1995-01-01

    The Naval Research Laboratory (NRL) Library and the American Physical Society (APS) are experimenting with electronically disseminating journals and reports in a project called TORPEDO (The Optical Retrieval Project: Electronic Documents Online). Scanned journals and reports are converted to ASCII, then attached to bibliographic information, and…

  20. TORPEDO: Networked Access to Full-Text and Page-Image Representations of Physics Journals and Technical Reports.

    ERIC Educational Resources Information Center

    Atkinson, Roderick D.; Stackpole, Laurie E.

    1995-01-01

    The Naval Research Laboratory (NRL) Library and the American Physical Society (APS) are experimenting with electronically disseminating journals and reports in a project called TORPEDO (The Optical Retrieval Project: Electronic Documents Online). Scanned journals and reports are converted to ASCII, then attached to bibliographic information, and…

  1. Changes in particular glycogen populations of the nerve terminals of Torpedo electric organ stimulated to fatigue in vitro.

    PubMed

    Constant, P; Goffinet, G; Schoffeniels, E

    1980-01-01

    Stimulation to fatigue of Torpedo electric organ fragments in partial anoxic conditions induces a loss of particular glycogen associated with the nerve terminals. These results show that the metabolic requirements imposed by the process of bioelectrogenesis in anoxia involve the utilisation glycogen.

  2. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

    PubMed

    Young, Gareth T; Zwart, Ruud; Walker, Alison S; Sher, Emanuele; Millar, Neil S

    2008-09-23

    Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

  3. Growth of the Obligate Anaerobe Desulfovibrio vulgaris Hildenborough under Continuous Low Oxygen Concentration Sparging: Impact of the Membrane-Bound Oxygen Reductases

    PubMed Central

    Ramel, Fanny; Brasseur, Gael; Pieulle, Laetitia; Valette, Odile; Hirschler-Réa, Agnès; Fardeau, Marie Laure; Dolla, Alain

    2015-01-01

    Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions

  4. The 3' untranslated region of the membrane-bound IL-1R accessory protein mRNA confers tissue-specific destabilization.

    PubMed

    Jensen, Liselotte E; Whitehead, Alexander S

    2004-11-15

    IL-1alpha and IL-1beta are proinflammatory cytokines that promote activation of intracellular signaling cascades, leading to stabilization of certain mRNAs and activation of transcription factors. IL-1R type I (IL-1RI) binds IL-1alpha and IL-1beta, and subsequent recruitment of the membrane-bound IL-1R accessory protein (mIL-1RAcP) facilitates signal transduction. Two alternatively spliced isoforms, soluble IL-1RAcP (sIL-1RAcP) and sIL-1RAcP-beta, which lack transmembrane and intracellular domains, have been described. The sIL-1RAcP and possibly sIL-1RAcP-beta can inhibit IL-1 signaling. Proportional expression of the different IL-1RAcP splice variants may be an important determinant of responsiveness to IL-1. We show that although both mIL-1RAcP and sIL-1RAcP mRNAs are widely expressed in human tissue, their relative proportions differ significantly in a tissue-specific manner. Turnover studies revealed that the sIL-1RAcP mRNA has a half-life of approximately 48 h in both the kidney cell line 293 and the hepatoma cell line HepG2. The mIL-1RAcP mRNA has a similar half-life in 293 cells, but a considerably shorter half-life of approximately 5 h in HepG2 cells. Using luciferase reporter constructs, we demonstrated that this specific destabilization of the mIL-1RAcP mRNA in the latter cell type is mediated by its 2.8-kb 3'-untranslated region. Deletion analysis further established that the cell line-specific instability does not involve AU-rich elements, but is mediated by several novel elements that appear to act independently; such elements may be recognized by proteins expressed specifically in some, but not all, tissues. These data demonstrate that the cellular capacity to respond to IL-1 is tightly regulated in a tissue-specific manner.

  5. Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases.

    PubMed

    Ramel, Fanny; Brasseur, Gael; Pieulle, Laetitia; Valette, Odile; Hirschler-Réa, Agnès; Fardeau, Marie Laure; Dolla, Alain

    2015-01-01

    Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions

  6. Membrane-Bound CYB5R3 Is a Common Effector of Nutritional and Oxidative Stress Response Through FOXO3a and Nrf2

    PubMed Central

    Siendones, Emilio; SantaCruz-Calvo, Sara; Martín-Montalvo, Alejandro; Cascajo, María V.; Ariza, Julia; López-Lluch, Guillermo; Villalba, José M.; Acquaviva-Bourdain, Cécile; Roze, Emmanuel; Bernier, Michel; de Cabo, Rafael

    2014-01-01

    Abstract Aims: Membrane-bound CYB5R3 deficiency in humans causes recessive hereditary methaemoglobinaemia (RHM), an incurable disease that is characterized by severe neurological disorders. CYB5R3 encodes for NADH-dependent redox enzyme that contributes to metabolic homeostasis and stress protection; however, how it is involved in the neurological pathology of RHM remains unknown. Here, the role and transcriptional regulation of CYB5R3 was studied under nutritional and oxidative stress. Results: CYB5R3-deficient cells exhibited a decrease of the NAD+/NADH ratio, mitochondrial respiration rate, ATP production, and mitochondrial electron transport chain activities, which were associated with higher sensitivity to oxidative stress, and an increase in senescence-associated β-galactosidase activity. Overexpression of either forkhead box class O 3a (FOXO3a) or nuclear factor (erythroid-derived 2)-like2 (Nrf2) was associated with increased CYB5R3 levels, and genetic ablation of Nrf2 resulted in lower CYB5R3 expression. The presence of two antioxidant response element sequences in the CYB5R3 promoter led to chromatin immunoprecipitation studies, which showed that cellular stressors enhanced the binding of Nrf2 and FOXO3a to the CYB5R3 promoter. Innovation: Our findings demonstrate that CYB5R3 contributes to regulate redox homeostasis, aerobic metabolism, and cellular senescence, suggesting that CYB5R3 might be a key effector of oxidative and nutritional stress pathways. The expression of CYB5R3 is regulated by the cooperation of Nrf2 and FOXO3a. Conclusion: CYB5R3 is an essential gene that appears as a final effector for both nutritional and oxidative stress responses through FOXO3a and Nrf2, respectively, and their interaction promotes CYB5R3 expression. These results unveil a potential mechanism of action by which CYB5R3 deficiency contributes to the pathophysiological underpinnings of neurological disorders in RHM patients. Antioxid. Redox Signal. 21, 1708–1725. PMID

  7. Degradation of membrane-bound ganglioside GM1. Stimulation by bis(monoacylglycero)phosphate and the activator proteins SAP-B and GM2-AP.

    PubMed

    Wilkening, G; Linke, T; Uhlhorn-Dierks, G; Sandhoff, K

    2000-11-17

    According to our hypothesis (Fürst, W., and Sandhoff, K. (1992) Biochim. Biophys. Acta 1126, 1-16) glycosphingolipids of the plasma membrane are digested after endocytosis as components of intraendosomal and intralysosomal vesicles and membrane structures. The lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acid exohydrolases requires small, non-enzymatic cofactors, called sphingolipid activator proteins (SAPs). A total of five activator proteins have been identified as follows: namely the saposins SAP-A, -B, -C, and -D, which are derived from the single chain SAP-precursor protein (prosaposin), and the GM2 activator protein. A deficiency of prosaposin results in the storage of ceramide and sphingolipids with short oligosaccharide head groups. The loss of the GM2 activator protein blocks the degradation of the ganglioside GM2. The enzymatic hydrolysis of the ganglioside GM1 is catalyzed by beta-galactosidase, a water-soluble acid exohydrolase. The lack of ganglioside GM1 accumulation in patients suffering from either prosaposin or GM2 activator protein deficiency has led to the hypothesis that SAPs are not needed for the hydrolysis of the ganglioside GM1 in vivo. In this study we demonstrate that an activator protein is required for the enzymatic degradation of membrane-bound ganglioside GM1 and that both SAP-B and the GM2 activator protein significantly enhance the degradation of the ganglioside GM1 by acid beta-galactosidase in a liposomal, detergent-free assay system. These findings offer a possible explanation for the observation that no storage of the ganglioside GM1 has been observed in patients with either isolated prosaposin or isolated GM2 activator deficiency. We also demonstrate that anionic phospholipids such as bis(monoacylglycero)phosphate and phosphatidylinositol, which specifically occur in inner membranes of endosomes and in lysosomes, are essential for the activator-stimulated hydrolysis of the ganglioside GM1

  8. Minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor

    SciTech Connect

    Jones, O.T.; Eubanks, J.H.; Earnest, J.P.; McNamee, M.G.

    1988-05-17

    The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

  9. Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes.

    PubMed

    Souroujon, M C; Barchan, D; Fuchs, S

    1985-01-01

    Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.

  10. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.

  11. Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture

    PubMed Central

    1987-01-01

    We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster. PMID:3793762

  12. Spectroscopic investigation of the nicotinic acetylcholine receptor for application in medical diagnosis

    NASA Astrophysics Data System (ADS)

    Salzer, Reiner; Fischer, Wolfgang B.; Unverricht, Ines; Schwenke, Dirk; Steiner, Gerald; Schrattenholz, Andre; Maelicke, Alfred

    1998-04-01

    Native vesicles containing the nicotinic acetylcholine receptor (nAChR) prepared from the electric organ of the ray Torpedo marmorata were used to obtain fluorescence signal sin dependence of different concentrations of the local anesthetics procaine. Nonlinear concentration dependent spectral changes are found using ethidium bromide as a fluorescence marker. Structural changes are found for the proteins including the nAChR in the vesicles during immobilization onto surfaces such as IR transparent germanium (GE) crystal, Ge crystal coated with silver (Ag) cluster to use the SEIRA effect and/or crystals covered with a lipid subphase. A comparison between Ge and Ge coated with Ag (Ge/Ag) clusters reveals increased structural changes in the spectral regions around 1670 cm-1 upon adsorption of the vesicles on the latter surface. Is the Ge/Ag crystal precoated with a lipid subphase an almost similar spectral contour for the amide I band envelope as in the spectra recorded on a neat Ge crystal is found.

  13. Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: Identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue

    PubMed Central

    Triquigneaux, Mathilde M.; Ehrenshaft, Marilyn; Roth, Esther; Silman, Israel; Ashani, Yakov; Mason, Ronald P.; Weiner, Lev; Deterding, Leesa J.

    2013-01-01

    Synopsis The principal role of acetylcholinesterase (AChE) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a catalytic ‘anionic’ subsite (CAS) and a second peripheral ‘anionic’ site (PAS), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophans (Trps). It was earlier shown that generation of 1O2 by illumination of methylene blue (MB) causes irreversible inactivation of Torpedo californica AChE (TcAChE), and suggested that photo-oxidation of Trps might be responsible. In the present study, structural modification of the TcAChE Trps induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and mass spectrometry. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp 84 and Trp 279 – present at the CAS and PAS, respectively. Peptides containing these two oxidized Trp residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus, the bound MB elicited selective action of 1O2 on Trp residues facing onto the water-filled active-site gorge. Our findings thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents. PMID:22888904

  14. An unusual phospholipase A₂ from puff adder Bitis arietans venom--a novel blocker of nicotinic acetylcholine receptors.

    PubMed

    Vulfius, Catherine A; Gorbacheva, Elena V; Starkov, Vladislav G; Osipov, Alexey V; Kasheverov, Igor E; Andreeva, Tatyana V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2011-04-01

    The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Isolation and characterization of two homologous cDNA clones from Torpedo electromotor neurons.

    PubMed

    Ngsee, J K; Scheller, R H

    1989-10-01

    Two homologous cDNA clones were isolated from a Torpedo california electric lobe lambda gt11 expression library using a polyclonal antiserum directed against proteins associated with synaptic vesicles. Northern blotting reveals an 8- to 9-kb transcript in the electric lobe and the spinal cord, but not in the brain or other non-neuronal tissues. Antibodies generated against a fusion protein synthesized in Escherichia coli reacted with a 85- to 90-kD species in the neurons of the electric lobe. The immunoreactivity is associated with microsomal membranes and can be extracted readily with high salt. Immunohistochemical studies demonstrated a sparse punctate staining pattern in the cell body which colocalized with a subpopulation of post-Golgi vesicles.

  16. Development of the electromotor system in Torpedo marmorata: cationic staining of the electric organ.

    PubMed

    Fox, G Q

    1987-10-01

    The electric organs of embryonic Torpedo marmorata have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neurites during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.

  17. Phospholipid turnover in Torpedo marmorata electric organ during discharge in vivo.

    PubMed Central

    Bleasdale, J E; Hawthorne, J N; Widlund, L; Heilbronn, E

    1976-01-01

    One electric organ of anaesthetized Torpedo marmorata was stimulated through electrodes placed on the electric lobe of the brain. Nerves to the other electric organ were cut to provide an unstimulated control. Glucose 6-[32P]phosphate was injected into each organ 16h before electrical stimulation. After stimulation for 10 min at 5 Hz, the organs were removed homogenized and centrifuged on a density gradient for the preparation of subcellular fractions. Stimulation increased the incorporation of 32P into phosphatidate, phosphatidylinositol and phosphatidylcholine. The increased phosphatidate labelling, but not that of the other two lipids, was seen in fractions rich in synaptic vesicles. Stimulation had no effect on ATP labelling. The phosphatidate content of most fractions fell slightly after stimulation, but amounts of other phospholipids were not affected. Images PLATE 1 PLATE 2 PMID:825114

  18. Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

    PubMed Central

    Mnatsakanyan, Nelli; Jansen, Michaela

    2013-01-01

    Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α-helical segments (M1–M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently-solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel α (GluClα)showed the same overall architecture . However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2–M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel (GLIC) and GluClα are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes. PMID:23565737

  19. The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

    PubMed Central

    Williamson, P. T. F.; Verhoeven, A.; Miller, K. W.; Meier, B. H.; Watts, A.

    2007-01-01

    The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali. The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >CO chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein. PMID:17989232

  20. Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides.

    PubMed Central

    Marinou, Martha; Tzartos, Socrates J

    2003-01-01

    The neuronal alpha7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin alpha-bungarotoxin (alpha-Bgt). Fine mapping of the alpha-Bgt-binding site on the human alpha7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human alpha7 subunit (residues 1-206). Screening of these peptides for (125)I-alpha-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186-197, which exhibited the best (125)I-alpha-Bgt binding, and one at residues 159-165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the alpha7(186-197) peptide was almost completely inhibited by unlabelled alpha-Bgt or d -tubocurarine. Alanine substitutions within the sequence 186-198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the alpha-Bgt binding site of the alpha1 subunit (residues 188-200 in Torpedo AChR). In competition experiments, the soluble peptides alpha7(186-197) and Torpedo alpha1(184-200) inhibited the binding of (125)I-alpha-Bgt to the immobilized alpha7(186-197) peptide, to native Torpedo AChR, and to the extracellular domain of the human alpha1 subunit. These results suggest that the toxin-binding sites of the neuronal alpha7 and muscle-type AChRs bind to identical or overlapping sites on the alpha-Bgt molecule. In support of this, when synthetic alpha-Bgt peptides were tested for binding to the recombinant extracellular domains of the human alpha7 and alpha1 subunits, and to native Torpedo and alpha7 AChR, the results indicated that alpha-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail. PMID:12614199

  1. Torpedo marmorata acetylcholinesterase; a comparison with the Electrophorus electricus enzyme. Molecular forms, subunits, electron microscopy, immunological relationship.

    PubMed

    Rieger, F; Bon, S; Massoulié, J; Cartauld, J; Picard, B; Benda, P

    1976-09-15

    Electron microscopy, sequential degradation by hydrolytic enzymes and the physical-chemical properties of the molecular forms of Torpedo acetylcholinesterase indicate that these molecules are structurally related to each other in the same way as the molecular forms of Electrophorus acetylcholinesterase: all are derived from a complex structure in which three tetrameric groups of subunits are associated with a rod-like 'tail'. In aged preparations the catalytic subunits are split into fragments in a manner similar to those of Electrophorus acetylcholinesterase. Immunological cross-reaction between both enzymes demonstrates the occurrence of common antigenic sites. The enzymes from the two sources, however, are different in their molecular weights and susceptibility to hydrolytic enzymes. Also, Torpedo acetylcholinesterase does not precipitate with either isologous or heterologous antibodies.

  2. Effect of denervation on a cholinergic-specific ganglioside antigen (Chol-1) present in Torpedo electromotor presynaptic plasma membranes.

    PubMed

    Ferretti, P; Borroni, E

    1984-04-01

    The presence of Chol-1, an antigen identified in the plasma membrane of cholinergic electromotor nerve terminals of Torpedo marmorata, was investigated in Torpedo electric organ after 3, 6, and 9 weeks' denervation. Denervation was monitored by the cessation of stimulus-evoked discharge potentials, by the reduction in nerve terminals seen morphologically, and by the decrease in ACh and ChAT contents. The content of ganglioside-bound sialic acid did not show any appreciable change with time. Some modification of ganglioside pattern on TLC was observed after 9 weeks' denervation. The presence of Chol-1 after denervation was assayed by its activity in inhibiting the selective complement-induced lysis of the cholinergic subpopulation of guinea pig cortical synaptosome which is mediated by the anti-Chol-1 antiserum. Denervation did not affect Chol-1 immunoreactivity although it did alter the distribution of the immunoreactivity among gangliosides. The possible significance of the results is discussed.

  3. Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain.

    PubMed Central

    Swanson, L W; Lindstrom, J; Tzartos, S; Schmued, L C; O'Leary, D D; Cowan, W M

    1983-01-01

    We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain. First, all of the mAbs that stained the SpL also stained neuromuscular junctions in skeletal muscle, whereas none of the 40 mAbs that failed to stain end plates crossreacted with the SpL; second, in vitro immunological studies and blocking experiments on tissue sections (in which unlabeled mAbs were used to block the staining of a directly fluorescein-treated mAb) indicated the presence of mAbs specific for unique antigenic determinants on all four of the subunits (alpha, beta, gamma, and delta) from Torpedo nAcChoR in chick midbrain and muscle. On the other hand, the distribution of mAb staining in the optic tectum does not closely parallel that of either acetylcholinesterase staining or of 125I-labeled alpha-bungarotoxin binding; no toxin binding has been observed autoradiographically in the SpL, but the nucleus does contain moderately dense acetylcholinesterase staining. Take together, our observations suggest that there may be a cholinergic input to the SpL and that the projection fibers from the SpL to the optic tectum (which are also stained with an antiserum to [Leu]enkephalin) may contain presynaptic nAcChoRs. It is clear, however

  4. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor.

    PubMed

    Lentz, T L

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of 125I-alpha-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b (IC50 = 5.7 x 10(-6) M) and the structurally similar segment (residues 173-203) of CVS rabies virus glycoprotein (IC50 = 2.6 x 10(-6) M). These affinities were comparable to those of d-tubocurarine (IC50 = 3.4 x 10(-6) M) and suberyldicholine (IC50 = 2.5 x 10(-6) M). These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Modifications involving Lys-27, Trp-29, Phe-33, Arg-37, and Gly-38 reduced affinity of binding. R37D and F33T modifications reduced the affinity of alpha-bungarotoxin residues 28-40 by an order of magnitude. Arg-37 may correspond to the positively charged quaternary ammonium group and Phe-33 to the hydrophobic acetyl methyl group of acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Acetylcholine functionally reorganizes neocortical microcircuits

    PubMed Central

    Runfeldt, Melissa J.; Sadovsky, Alexander J.

    2014-01-01

    Sensory information is processed and transmitted through the synaptic structure of local cortical circuits, but it is unclear how modulation of this architecture influences the cortical representation of sensory stimuli. Acetylcholine (ACh) promotes attention and arousal and is thought to increase the signal-to-noise ratio of sensory input in primary sensory cortices. Using high-speed two-photon calcium imaging in a thalamocortical somatosensory slice preparation, we recorded action potential activity of up to 900 neurons simultaneously and compared local cortical circuit activations with and without bath presence of ACh. We found that ACh reduced weak pairwise relationships and excluded neurons that were already unreliable during circuit activity. Using action potential activity from the imaged population, we generated functional wiring diagrams based on the statistical dependencies of activity between neurons. ACh pruned weak functional connections from spontaneous circuit activations and yielded a more modular and hierarchical circuit structure, which biased activity to flow in a more feedforward fashion. Neurons that were active in response to thalamic input had reduced pairwise dependencies overall, but strong correlations were conserved. This coincided with a prolonged period during which neurons showed temporally precise responses to thalamic input. Our results demonstrate that ACh reorganizes functional circuit structure in a manner that may enhance the integration and discriminability of thalamic afferent input within local neocortical circuitry. PMID:24872527

  6. Palmitoylation of Nicotinic Acetylcholine Receptors

    PubMed Central

    Alexander, J. K.; Govind, A. P.; Drisdel, R. C.; Blanton, M. P.; Vallejo, Y.; Lam, T. T.

    2012-01-01

    It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different post-translational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal “α4β2” and “α7” subtypes most abundant in brain. The loss of ligand binding sites that occurs when palmitoylation is blocked with the inhibitor bromopalmitate suggests that palmitoylation of α4β2 and α7 subtypes occurs during subunit assembly and regulates the formation of ligand binding sites. However, additional experiments are needed to test whether nAChR subunit palmitoylation is involved in other aspects of nAChR trafficking or whether palmitoylation regulates nAChR function. Further investigation would be aided by identifying the sites of palmitoylation on the subunits, and here we propose a mass spectrometry strategy for identification of these sites. PMID:19693711

  7. Determination By X-Ray Crystallography of the Three Dimensional- Structure of Acetylcholinesterase from Torpedo Electric Organ

    DTIC Science & Technology

    1993-04-17

    organ tissue of Electrophorus electricus (27). Although preliminary characterization of these crystals was reported over 25 years ago by Chothia...The different oligomeric forms of AChE in the electric organ of the electric fish, Electrophorus and Torpedo, are structurally homologous to those in...attached phosphatidylinositol (PI) (10). This dirmer has a simpler quaternary structure than Electrophorus AChE, and its sequence and the arrangement of

  8. Determination by X-Ray Crystallography of the Three-Dimensional Structure of Acetylcholinesterase from Torpedo Electric Organ

    DTIC Science & Technology

    1990-10-01

    from Electrophorus electricus was first crystallized by Leuzinger and Baker (1967), and preliminary characterization of crystals obtained from this form...to organophosphorus poisoning. The electric organs of the electric fish Torpedo and Electrophorus provide rich sources of AChE (Nachmansohn and...was subsequently reported (Chothia and Leuzinger, 1975; Schrag et al., 1988). Sequence information concerning Electrophorus AChE is not yet available

  9. Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

    PubMed

    Padilla-Morales, Luis F; Colón-Sáez, José O; González-Nieves, Joel E; Quesada-González, Orestes; Lasalde-Dominicci, José A

    2016-01-01

    In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex

  10. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer.

  11. [The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

    PubMed

    Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

    2016-01-01

    Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

  12. Torpedo electromotor system development: developmentally regulated neuronotrophic activities of electric organ tissue.

    PubMed

    Richardson, G P; Rinschen, B; Fox, G Q

    1985-01-15

    Explant cultures of electric lobe from 45-60 mm stage Torpedo embryos and both ganglionic and dissociated cell cultures prepared from 8-day chick ciliary ganglia have been used to determine whether the electric organs of Torpedo marmorata contain developmentally regulated neuronotrophic activity. Electric lobe explants were evaluated by measuring their neurone density, choline acetyltransferase (CAT0, and low salt, Triton X-100-soluble protein contents. Addition of soluble extracts prepared from the electric organs of late stage embryos (85-105 mm) to standard medium results in the maintenance of nearly theoretical neurone densities in electric lobe explants during a 7-day culture period. Soluble electric organ extracts from early embryonic stages (42-59 mm) do not increase neurone density relative to control cultures but cause an elevation in the CAT content of the explants over control values. On the basis of this analysis it is concluded (1) that late embryonic stage and adult electric organs contain neuronotrophic activity that allows electromotor neurones to survive in vitro and (2) that activity increases rapidly in the electric organs between the 59 nd 72 mm stages of development at a time when rapid increases in postsynaptic membrane markers in the electric organs occur and when peripheral synaptogenesis begins. The activity of late stage embryonic electric organs is heat stable and lost on dialysis. Using ciliary ganglion explants and evaluating both the initial fibre outgrowth and the CAT content after 4 days in vitro, trophic activity is found to be maximal at early embryonic stages (45-55 mm) and to decline thereafter. It is shown that the decline in activity is not due to an increase in toxicity. Using established dissociated ganglionic cell survival assays the specific activity of neuronotrophic factors allowing survival is constant between the 45 and 73 mm stages in the electric organs and then rapidly declines, but activity per electric organ

  13. Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Valera, S.; Ballivet, M.; Bertrand, D.

    1992-10-01

    The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

  14. Presence of vasoactive intestinal polypeptide-like immunoreactivity in the cholinergic electromotor system of Torpedo marmorata.

    PubMed

    Agoston, D V; Conlon, J M

    1986-08-01

    Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was detected in the cholinergic electro-motor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP-like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle-specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP-like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (-50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP-like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse-phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP-like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.

  15. 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

    SciTech Connect

    Carr, C.; McCourt, D.; Cohen, J.B.

    1987-11-03

    The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The /sup 14/C-labelled 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH/sub 2/-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approx. 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic ..cap alpha..-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.

  16. Metabolism of acetylcholine in human erythrocytes

    SciTech Connect

    Chapman, E.S.

    1990-01-01

    In order to examine the possible role of erythrocyte acetylcholinesterase in the maintenance of membrane phospholipid content and membrane fluidity, experiments were performed to monitor the activity of the enzyme and follow the fate of one of its hydrolytic products, choline. Intact human erythrocytes were incubated with acetylcholine (choline methyl-{sup 14}C). The incubation resulted in the hydrolysis of acetylcholine to acetate and choline; the reaction was catalyzed by membrane acetylcholinesterase. The studies demonstrate the further metabolism of choline. Experiments were carried out to determine rate of hydrolysis of acetylcholine, uptake of choline, identification of intracellular metabolites of choline, and identification of radiolabeled membrane components. Erythrocytes at a 25% hematocrit were incubated in an isoosmotic bicarbonate buffer pH 7.4, containing glucose, adenosine, streptomycin and penicillin with 0.3 {mu}Ci of acetylcholine (choline methyl-{sup 14}C), for 24 hours. Aliquots of the erythrocyte suspension were taken throughout for analysis. Erythrocytes were washed free of excess substrate, lysed, and the hemolysate was extracted for choline and its metabolites. Blank samples containing incubation buffer and radiolabeled acetylcholine only, and erythrocyte hemolysate extracts were analyzed for choline content, the difference between blank samples and hemolysate extracts was the amount of choline originating from acetylcholine and attributable to acetylcholinesterase activity. The conversion of choline to {sup 14}C-betaine is noted after several minutes of incubation; at 30 minutes, more than 80% of {sup 14}C-choline is taken up and after several hours, detectable levels of radiolabeled S-adenosylmethionine were present in the hemolysate extract.

  17. Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor.

    PubMed

    Lentz, T L; Hawrot, E; Wilson, P T

    1987-01-01

    Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha

  18. Surplus acetylcholine and acetylcholine release in the rat diaphragm.

    PubMed Central

    Molenaar, P C; Oen, B S; Polak, R L; van der Laaken, A L

    1987-01-01

    1. Skeletal muscles from rat, mouse and frog were incubated under different conditions and the amounts of acetylcholine (ACh) extractable from the tissue and released into the medium were determined by mass fragmentography. In some experiments measurements were made of the amounts of ACh ('bound' ACh) surviving in a muscle homogenate to which an excess of acetylcholinesterase had been added. In other experiments the membrane potentials, end-plate potentials (e.p.p.s), and miniature end-plate potentials (m.e.p.p.s) were studied. 2. During incubation in Ringer medium the ACh content of the rat hemidiaphragm usually did not change, but after inhibition of cholinesterase by soman the ACh content rose gradually from about 100 to 150 pmol to a plateau of about 400 pmol after 4 h. A similar formation of 'surplus ACh' after cholinesterase inhibition was found in the mouse diaphragm, but not in the frog sartorius muscle. 3. Surplus ACh accumulated predominantly in the end-plate region of the rat diaphragm. In muscles, 16-18 h after in vivo denervation, the capacity to form surplus ACh was decreased by more than 80%. 4. The amount of ACh diffusing from the resting hemidiaphragm into the incubation medium ('resting release') varied between 0.5 and 0.9 pmol min-1 in different experiments; it remained at the same level during accumulation of surplus ACh. It was reduced by more than 80% 16-18 h after denervation. 5. The amplitude of m.e.p.p.s and e.p.p.s did not increase while surplus ACh was accumulating. 6. Incubation of hemidiaphragms in Ringer solution containing [3H]choline caused the formation of [3H]ACh. Additional amounts of [3H]choline were incorporated into ACh when the nerve was stimulated for 60 min. However, incubation in the presence of soman (3,3-dimethyl-2-butylmethylphosphonofluoridate), in the absence of stimulation, did not cause an increase of the [3H]ACh content of the muscles. 7. From hemidiaphragms with active cholinesterase about 120 pmol ACh was lost

  19. Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

    PubMed

    Sivaprakasam, Kannan; Pagán, Oné R; Hess, George P

    2010-02-01

    Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10(11) neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.

  20. Downstream coding region determinants of bacterio-opsin, muscarinic acetylcholine receptor and adrenergic receptor expression in Halobacterium salinarum.

    PubMed

    Bartus, Cynthia L; Jaakola, Veli-Pekka; Reusch, Regina; Valentine, Helene H; Heikinheimo, Pirkko; Levay, Agata; Potter, Lincoln T; Heimo, Heikki; Goldman, Adrian; Turner, George J

    2003-02-17

    The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum. Copyright 2003 Elsevier Science B.V.

  1. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor. alpha. subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    SciTech Connect

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M. )

    1991-05-21

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.

  2. Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

    PubMed Central

    Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds. PMID:26008231

  3. Structure-activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states.

    PubMed

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2011-09-01

    The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca²⁺ influx in human embryonic muscle AChRs with the following potency sequence (IC(50) in μM): (±)-18-methylaminocoronaridine (5.9±0.3)∼(±)-18-methoxycoronaridine (18-MC) (6.8±0.8)>(-)-ibogaine (17±3)∼(+)-catharanthine (20±1)>(±)-albifloranine (46±13), (b) bind to the [³H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [³H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [³H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC₅₀) that correlate very well with the pK(i) values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 ų for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization.

  4. Turnover of Acetylcholine Receptors: Mechanisms of Regulation

    DTIC Science & Technology

    1988-12-01

    ME, Whittingham S, and Duane DD (1976) Antibody to acetylcholine receptor in myasthenia gravis : prevalance, clinical correlates and diagnostic value...transferred to nitorcellulose. Proc Natl Acad Sci 77:5201-5205. Weinberg CB and Hall ZW (1979) Antibodies from patients with myasthenia gravis recognize

  5. Nicotinic acetylcholine receptor from chick optic lobe.

    PubMed Central

    Norman, R I; Mehraban, F; Barnard, E A; Dolly, J O

    1982-01-01

    An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors. Images PMID:6175967

  6. Primary Structure of Nicotinic Acetylcholine Receptor

    DTIC Science & Technology

    1986-08-01

    quantities of starting material (for reviews of receptor, see Popot and Changeux, 1984; Stroud and Finer-Moore, 1985). This work led to the...Cloning of the Acetylcholine Receptor. Cold Spring Harbor Symp. on Quant. Biol. XLVIH: 71-78. 15. Popot , J-L. and Changeux, J-P. (1984) The

  7. Molecular dynamics simulation of the M2 helices within the nicotinic acetylcholine receptor transmembrane domain: structure and collective motions.

    PubMed

    Hung, Andrew; Tai, Kaihsu; Sansom, Mark S P

    2005-05-01

    Multiple nanosecond duration molecular dynamics simulations were performed on the transmembrane region of the Torpedo nicotinic acetylcholine receptor embedded within a bilayer mimetic octane slab. The M2 helices and M2-M3 loop regions were free to move, whereas the outer (M1, M3, M4) helix bundle was backbone restrained. The M2 helices largely retain their hydrogen-bonding pattern throughout the simulation, with some distortions in the helical end and loop regions. All of the M2 helices exhibit bending motions, with the hinge point in the vicinity of the central hydrophobic gate region (corresponding to residues alphaL251 and alphaV255). The bending motions of the M2 helices lead to a degree of dynamic narrowing of the pore in the region of the proposed hydrophobic gate. Calculations of Born energy profiles for various structures along the simulation trajectory suggest that the conformations of the M2 bundle sampled correspond to a closed conformation of the channel. Principal components analyses of each of the M2 helices, and of the five-helix M2 bundle, reveal concerted motions that may be relevant to channel function. Normal mode analyses using the anisotropic network model reveal collective motions similar to those identified by principal components analyses.

  8. Guidelines for standard preclinical experiments in the mouse model of myasthenia gravis induced by acetylcholine receptor immunization.

    PubMed

    Tuzun, Erdem; Berrih-Aknin, Sonia; Brenner, Talma; Kusner, Linda L; Le Panse, Rozen; Yang, Huan; Tzartos, Socrates; Christadoss, Premkumar

    2015-08-01

    Myasthenia gravis (MG) is an autoimmune disorder characterized by generalized muscle weakness due to neuromuscular junction (NMJ) dysfunction brought by acetylcholine receptor (AChR) antibodies in most cases. Although steroids and other immunosuppressants are effectively used for treatment of MG, these medications often cause severe side effects and a complete remission cannot be obtained in many cases. For pre-clinical evaluation of more effective and less toxic treatment methods for MG, the experimental autoimmune myasthenia gravis (EAMG) induced by Torpedo AChR immunization has become one of the standard animal models. Although numerous compounds have been recently proposed for MG mostly by using the active immunization EAMG model, only a few have been proven to be effective in MG patients. The variability in the experimental design, immunization methods and outcome measurements of pre-clinical EAMG studies make it difficult to interpret the published reports and assess the potential for application to MG patients. In an effort to standardize the active immunization EAMG model, we propose standard procedures for animal care conditions, sampling and randomization of mice, experimental design and outcome measures. Utilization of these standard procedures might improve the power of pre-clinical EAMG experiments and increase the chances for identifying promising novel treatment methods that can be effectively translated into clinical trials for MG.

  9. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

    USGS Publications Warehouse

    Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

    2014-01-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

  10. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  11. The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf.

    PubMed

    Meyer, Birte; Kuehl, Jennifer V; Price, Morgan N; Ray, Jayashree; Deutschbauer, Adam M; Arkin, Adam P; Stahl, David A

    2014-11-01

    The adaptation capability of Desulfovibrio to natural fluctuations in electron acceptor availability was evaluated by studying Desulfovibrio alaskensis strain G20 under varying respiratory, fermentative and methanogenic coculture conditions in chemostats. Transition from lactate to pyruvate in coculture resulted in a dramatic shift in the population structure and closer interspecies cell-to-cell interactions. Lower methane production rates in coculture than predicted from pyruvate input was attributed to redirection of electron flow to fumarate reduction. Without a methanogenic partner, accumulation of H₂and formate resulted in greater succinate production. Comparative transcript and gene fitness analysis in concert with physiological data of G20 wildtype and mutants demonstrated that pyruvate fermentation involves respiration of cytoplasmically formed fumarate using cytoplasmic and membrane-bound energy-conserving complexes, Rnf, Hdr-Flox-1 and Hmc. At the low H₂/formate levels maintained in coculture, Rnf likely functions as proton-pumping ferredoxin (Fd): type-I cytochrome c oxidoreductase, which transitions to a proton-pumping Fd(red):  nicotinamide adenine dinucleotide (NAD⁺) oxidoreductase at high H₂/formate levels during fermentation in monoculture. Hdr-Flox-1 is postulated to recycle Fd(red) via a flavin-based electron bifurcation involving NADH, Fdox and the thiol/disulphide-containing DsrC. In a menaquinone (MQ)-based electron confurcation reaction, the high-molecular-weight cytochrome-c₃complex, Hmc, is proposed to then couple DsrC(red) and periplasmic H₂/formate oxidation using the MQ pool to fuel a membrane-bound fumarate reductase.

  12. Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma subunits of heterotrimeric G-proteins.

    PubMed Central

    Le Stunff, H; Dokhac, L; Bourgoin, S; Bader, M F; Harbon, S

    2000-01-01

    Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins. PMID:11085943

  13. Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site.

    PubMed

    Wangler, Naomi J; Santos, Kira L; Schadock, Ines; Hagen, Fred K; Escher, Emanuel; Bader, Michael; Speth, Robert C; Karamyan, Vardan T

    2012-01-02

    Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II