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Sample records for membrane-bound torpedo acetylcholine

  1. Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine

    SciTech Connect

    White, B.J.; Cohen, J.B.

    1988-11-29

    The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the ..gamma..-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist ..cap alpha..-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effect on (/sup 125/I)TID labeling of the AChR. The regions of the AChR ..cap alpha..-subunit that incorporate (/sup 125/)TID were mapped by Staphylococcus aureus V8 protest digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated (/sup 125/I)TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the ..cap alpha..-subunit amino terminus, incorporated no detectable (/sup 125/I)TID. The mapping results place constraints on suggested models of AChR subunit topology.

  2. Immunisation with Torpedo acetylcholine receptor.

    PubMed

    Elfman, L

    1984-01-01

    Acetylcholine mediates the transfer of information between neurons in the electric organ of, for example, Torpedo as well as in vertebrate skeletal muscle. The nicotinic acetylcholine receptor complex translates the binding of acetylcholine into ion permeability changes. This leads to an action potential in the muscle fibre. The nicotinic acetylcholine receptor protein has been purified from Torpedo by use of affinity chromatography. The receptor is an intrinsic membrane glycoprotein composed of five polypeptide chains. When various animals are immunised with the receptor they demonstrate clinical signs of severe muscle weakness coincident with high antibody titres in their sera. The symptoms resemble those found in the autoimmune neuromuscular disease myasthenia gravis in humans. This animal model has constituted a unique model for studying autoimmune diseases. This paper reviews some of the work using Torpedo acetylcholine receptor in order to increase the understanding of the motor nervous system function and myasthenia gravis. It is now known that the nicotinic acetylcholine receptor protein is the antigen involved in myasthenia gravis. The mechanism of immune damage involves a direct block of the receptor function. This depends on the presence of antibodies which crosslink the postsynaptic receptors leading to their degradation. The questions to be answered in the future are; (a) what initiates or triggers the autoimmune response, (b) how do the antibodies cause the symptoms--is there a steric hindrance of the interaction of acetylcholine and the receptor, (c) why is there not a strict relationship between antibody titre and severity of symptoms, and (d) why are some muscles affected and other spared? With help of the experimental model, answers to these questions may result in improved strategies for the treatment of the autoimmune disease myasthenia gravis. PMID:6097937

  3. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    PubMed

    Rozman, Klara Bulc; Araoz, Romulo; Sepcić, Kristina; Molgo, Jordi; Suput, Dusan

    2010-09-01

    Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR (alpha1(2)betagammadelta) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine (d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR. PMID:20230806

  4. Topographical studies of the nicotinic acetylcholine receptor. [Torpedo californica

    SciTech Connect

    Middlemas, D.S.

    1987-01-01

    All four subunits of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with the photoactivated hydrophobic probe, (/sup 3/H)adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. Since the photogenerated carbene is situated near the lipid-water interface, this probe has potential as a topographic tool for mapping membrane protein structure. The labeling studies with both (/sup 3/H)adamantanediazirine and (/sup 3/H)cholesteryl diazoacetate support the concept that the acetylcholine receptor is a pseudosymmetric complex of homologous subunits, all of which interact with and span the membrane. The synthesis of the fluorine-containing agonists for the Torpedo californica nicotinic acetylcholine receptor, fluoroacetylcholine bromide and p-fluorophenyltrimethylammonium iodide, are described. It is demonstrated that both are agonists using a cation flux assay with acetylcholine receptor enriched membrane vesicles. The affinity cleavage reagent, p-thiocyanophenyltrimethylammonium iodide, specifically cleaves a peptide bond of the nicotinic acetylcholine receptor in membrane vesicles isolated from Torpedo californica. It is demonstrated that this reagent is an agonist using a cation flux assay. The cleavage is blocked by stoichiometric quantities of ..cap alpha..-bungarotoxin.

  5. 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.

  6. 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.

  7. Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Miledi, R

    1989-01-01

    Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensitized as slowly as cat AcChoR. In contrast, the hybrid AcChoRs expressed by coinjection with the Torpedo gamma subunit mRNA desensitized much more rapidly than cat AcChoR. The AcChoRs expressed in oocytes injected with cat muscle mRNA together with the Torpedo beta, gamma, and delta subunit mRNAs desensitized as rapidly as Torpedo AcChoR, indicating that the cat alpha subunit does not play an important role in determining the slow rate of desensitization. It is concluded that the difference in the rates of desensitization of cat and Torpedo AcChoRs is determined mainly by differences in their respective gamma subunits. Images PMID:2536157

  8. Amino acids of the Torpedo marmorata acetylcholine receptor. cap alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

    SciTech Connect

    Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.Y.; Lazure, C.; Chretien, M.; Changeux, J.P.

    1988-04-05

    The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the ..cap alpha..-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment ..cap alpha.. 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the ..cap alpha..-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the ..cap alpha..-chain that assign the amino-terminal segment ..cap alpha.. 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified (/sup 3/H)DDF-labeled resides, which are conserved in muscle and neuronal ..cap alpha..-chains but not in the other subunits, may be directly involved in agonist binding.

  9. 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.

  10. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

    PubMed

    Hartig, P R; Raftery, M A

    1979-04-01

    Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes. PMID:427105

  11. Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

    PubMed Central

    Tank, D W; Huganir, R L; Greengard, P; Webb, W W

    1983-01-01

    Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the alpha 2 beta gamma delta subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels. PMID:6308673

  12. Mechanism of phencyclidine binding to the acetylcholine receptor from Torpedo electroplaque.

    PubMed

    Oswald, R E; Bamberger, M J; McLaughlin, J T

    1984-05-01

    The mechanism of phencyclidine binding to Torpedo acetylcholine receptor-rich membranes was investigated. The rate of [3H]phencyclidine association is 10(3)- to 10(4)-fold more rapid when phencyclidine and carbamoylcholine are added simultaneously to acetylcholine receptor-rich membranes than when phencyclidine is added to membranes previously equilibrated with carbamoylcholine or membranes in the absence of carbamoylcholine. The mechanism of binding under conditions in which the slower rate was observed was studied with thermodynamic, viscosity, and kinetic experiments. Association and dissociation rates were highly dependent on temperature with activation energies of 26-30 kcal/mole. Viscosity had no effect on the association rate but increased the dissociation rate. These studies suggest that the binding is not diffusion-controlled but rather is limited by a significant energy barrier. The association rate was determined as a function of the concentration of acetylcholine receptor-rich membranes and the concentration of phencyclidine. In the presence of carbamoylcholine, the association rate was highly dependent upon the concentration of acetylcholine receptor but virtually insensitive to the concentration of phencyclidine. In the absence of carbamoylcholine, the association rate seemed to be a hyperbolic function of both the phencyclidine and the acetylcholine receptor concentration. The minimal model capable of explaining the data is a mechanism by which phencyclidine binds to two conformations of the acetylcholine receptor, one conformation having a higher affinity and constituting a lower percentage of receptors and the other having a lower affinity and constituting a higher percentage. The data are consistent with the possibility that the high-affinity conformation is the open-channel state of the acetylcholine receptor. PMID:6727862

  13. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    SciTech Connect

    Forman, S.A.; Miller, K.W. )

    1989-02-21

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid {sup 86}Rb{sup +} quenched-flux assays at 4 {degree}C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with {alpha}-bungarotoxin ({alpha}-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k{sub d} observed without procaine. The dependence of k{sub r} on (procaine) is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of {alpha}-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k{sub r}'s dependence on (ACh) in channel-activating range closely parallels that of {sup 86}Rb{sup +} flux response to ACh.

  14. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

    PubMed Central

    Hucho, F; Layer, P; Kiefer, H R; Bandini, G

    1976-01-01

    Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (alpha), 48,000 (beta), 62,000 (gamma), and 66,000 (delta). The alpha and beta chains are still present in all and gamma and delta in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the delta chain incorporating less of the reagent as compared to the alpha and beta chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the alpha chain only. We conclude that the alpha chain binds the neurotoxin from Naja naja, the alpha and beta chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the gamma and delta chains remains to be determined. Images PMID:1066671

  15. Different channel properties of Torpedo acetylcholine receptor monomers and dimers reconstituted in planar membranes.

    PubMed Central

    Schindler, H; Spillecke, F; Neumann, E

    1984-01-01

    It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different. The native dimer D of Mr 500,000 (heavy-form) exhibits a "single" channel conductance about twice as large as that of the monomer M of Mr 250,000 (light form). Under conditions where monomers aggregate, the conductance changes from the level of the monomer M to that of dimers M2. The dimer conductances (D and M2) seem to result from synchronous opening and closing of the two channels in the dimer, giving the impression of "single channel" activity. This channel cooperativity is apparently mediated by noncovalent interactions between the two monomers, since it requires no disulfide linkage between monomers. Both the monomers M and the dimers D and M2 show at least one substate of lower conductivity. The relative population of the two conductance levels depends on the ion type (Na+ and K+), indicating ion-specific channel states. Since the channel conductance of isolated dimers resembles those obtained from unextracted microsacs, the dimer with two synchronized channels appears to be the in vivo predominant gating unit. In the linear association of dimers, observed in the native membrane, channel synchronization may extend to more than two channels as suggested by oligomeric channel cooperativity in associations of monomers and dimers. PMID:6091143

  16. Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

    SciTech Connect

    Dowding, A.J.; Hall, Z.W.

    1987-10-06

    The authors have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of ..cap alpha..-bungarotoxin (..cap alpha..-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block ..cap alpha..-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit ..cap alpha..-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds ..cap alpha..-BuTx with a forward rate constant of 0.98 x 10/sup 5/ M/sup -1/ s/sup -1/, d-tubocurarine (dTC) with a K/sub D/ of (6.8 +/- 0.3) x 10/sup -8/ M, and pancuronium with a K/sub D/ of (1.9 +/- 1.0) x 10/sup -9/ M. The site recognized by B mAbs binds ..cap alpha..-BuTx with a forward rate constant of 9.3 x 10/sup 5/ M/sup -1/ s/sup -1/, dTC with a K/sub D/ of (4.6 +/- 0.3) x 10/sup -6/ M, and pancurionium with a K/sub D/ of (9.3 +/- 0.8) x 10/sup -6/ M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by ..cap alpha..-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.

  17. Acetylcholine release evoked by single or a few nerve impulses in the electric organ of Torpedo.

    PubMed Central

    Dunant, Y; Eder, L; Servetiadis-Hirt, L

    1980-01-01

    1. The acetylcholine (ACh) store in the Torpedo electric organ was partially labelled with choline and acetate at the same molar concentration but with different isotopes. Under these conditions the two precursors were incorporated into ACh in a ratio 1 to 1. 2. After a single electrical stimulus, or a brief burst of stimuli, the compound electroplaque potential (e.p.p.) was recorded and the radioactive choline and/or acetate counted in the perfusion fluid, providing a sensitive assay for ACh release in the absence of anticholinesterase drugs. 3. The so-called depression of transmission was found to be due to progressive impairment of ACh release in the successive impulses evoked by repeated stimuli. 4. In a pair of impulses separated by 50 ms interval, less ACh was released by the second than by the first impulse; this explained why the size of the second e.p.p. was depressed, using a direct measurement of ACh. 5. In repetitive stimulations of longer duration, the maximum rate of release declined as the activity was prolonged. Thus the tissue progressively lost its ability to ensure release at high frequencies. 6. An unexpected finding was that anticholinesterases like eserine or pre-treatment with fluostigmine (DFP) greatly reduced ACh release even by a single impulse. 7. Evoked ACh release and e.p.p. amplitude were both maximum between 10 and 20 degrees C. At higher temperatures, the evoked release decreased as the spontaneous release increased. 8. Changes in external Ca2+ and Mg2+ produced similar changes in the e.p.p. and evoked ACh release. The dose--response curve for Ca dependency of ACh release was very steep with a Hill's coefficient of 3.2. 9. With a single stimulus in the presence of 4-aminopyridine, there was a dramatic enlargement of the e.p.p. and a still larger potentiation of the evoked ACh release. 10. It has been possible with this approach to avoid the inconveniences often encountered in simliar studies, i.e. repetitive stimulation, low Ca

  18. Pulsatile release of acetylcholine by nerve terminals (synaptosomes) isolated from Torpedo electric organ.

    PubMed Central

    Girod, R; Eder-Colli, L; Medilanski, J; Dunant, Y; Tabti, N; Poo, M M

    1992-01-01

    1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare

  19. 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.

  20. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

    PubMed

    Hamouda, Ayman K; Stewart, Deirdre S; Husain, S Shaukat; Cohen, Jonathan B

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

  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. 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 100μM 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. PMID:26851639

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

    PubMed Central

    Saitoh, T; Changeux, J P

    1981-01-01

    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. Images PMID:6945595

  5. 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

  6. Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

    PubMed Central

    Montal, M; Labarca, P; Fredkin, D R; Suarez-Isla, B A

    1984-01-01

    The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing. PMID:6324900

  7. Effects of CI-1002 and CI-1017 on spontaneous synaptic activity and on the nicotinic acetylcholine receptor of Torpedo electric organ.

    PubMed

    Ros, E; Aleu, J; Marsal, J; Solsona, C

    2000-02-25

    The effect of azepino[2,1-b]quinazoline 1,3-dichloro-6,7,8,9,10, 12-hexahydro-, mono-hydrochloride (CI-1002), a tacrine derivative, and 1-azabicyclo[2.2.1]heptan-3-one, O-[3-(methoxyphenyl)-2-propynyl]oxime [R-(Z)]-2-butenedioate (CI-1017), a muscarinic M(1) receptor agonist, on spontaneous synaptic activity was investigated by measuring amplitude, rise time, velocity of rising, half-width, and electrical charge of miniature endplate potentials (m.e.p.p.) recorded extracellularly in Torpedo electric organ fragments. The effect of CI-1002 and CI-1017 on the nicotinic acetylcholine receptor was investigated by measuring the current induced by acetylcholine in Xenopus laevis oocytes transplanted with membranes from Torpedo electric organ. CI-1002, at a concentration of 1 microM, altered the m.e.p.p. by increasing the amplitude (from 1.08+/-0.01 to 2.76+/-0.03 mV), rise time (from 0. 700+/-0.006 to 1.02+/-0.01 ms), rising rate (from 1.79+/-0.02 to 3. 45+/-0.05 mV/ms), half-width (from 0.990+/-0.008 to 2.40+/-0.02 ms), and electrical charge (from 304+/-4 to 784+/-11 mV s). CI-1017, at a concentration of 1 microM, altered the m.e.p.p. by decreasing the amplitude (from 1.08+/-0.01 to 0.650+/-0.007 mV), rise time (from 0. 700+/-0.006 to 0.530+/-0.007 ms), rising rate (from 1.79+/-0.02 to 1. 53+/-0.02 mV/ms), half-width (from 0.990+/-0.008 to 0.670+/-0.007 ms), and electrical charge (from 304+/-4 to 75+/-1 mV s). CI-1002 inhibited the acetylcholine-induced current of nicotinic acetylcholine receptors with an IC(50) of 3.4+/-0.3 microM. CI-1017 inhibited the acetylcholine-induced current of nicotinic acetylcholine receptors with an IC(50) of 0.8+/-0.1 microM. These results indicate that, although both drugs interacted negatively with nicotinic acetylcholine receptors, CI-1002 overcame this inhibition by recruiting more acetylcholine to build a quantum. PMID:10708701

  8. 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

  9. Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

    PubMed Central

    Wilson, P T; Gershoni, J M; Hawrot, E; Lentz, T L

    1984-01-01

    Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor. Images PMID:6371817

  10. Fluidity of the lipids next to the acetylcholine receptor protein of torpedo membrane fragments. Use of amphiphilic reversible spin-labels.

    PubMed

    Bienvenüe, A; Rousselet, A; Kato, G; Devaux, P F

    1977-03-01

    Choline esters of spin-labeled fatty acids (long-chain acylcholines) were used to probe the hydrophobic environment of the acetylcholine receptor protein in membrane fragments from Torpedo marmorata. These spin-labels competitively inhibit the binding of [3H]acetylcholine to the receptor site. Their inhibition constants (KI) were close to 200 nM. At the high membrane concentration required for electron spin resonance (ESR) experiments, the apparent inhibition constants (KIapp) differed from KI determined by using dilute membrane concentration. This is due to the amphiphilic character of long-chain acylcholine. For most spin-labels used, only difference ESR spectroscopy provided reliable spectra corresponding to receptor-bound spin-labeled acylcholines. Acetylcholine receptor agonists and antagonists displaced the acylcholine from the receptor sites, whereas choline had only a weak effect. This produced a modification in the ESR spectra of the bound acylcholines and provided evidence that the acylcholines bound to the receptor sites in a specific manner. The interpretation of the spectra of receptor-bound spin-labels favored a strong barrier to the motion of the probe when attached to the middle of the acyl chain. However, when the probe was close to the methyl terminal of a stearoylcholine molecule a much greater fluidity was found. Short-range spin-spin interactions were created between spin-labels bound to the receptor site and spin-labels in a fluid phase. This indicates that lipids next to the receptor protein are not completely immobilized in spite of the semicrystalline organization of the proteins in the postsynaptic region. PMID:191058

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

    PubMed

    Heuser, J E; Salpeter, S R

    1979-07-01

    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

  12. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  13. 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

  14. 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

    2012-12-01

    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

  15. 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.

  16. Domain Organization of Membrane-Bound Factor VIII

    PubMed Central

    Stoilova-McPhie, Svetla; Lynch, Gillian C.; Ludtke, Steven; Pettitt, B. Montgomery

    2014-01-01

    Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. PMID:23616213

  17. Myosin motors fragment and compact membrane-bound actin filaments

    PubMed Central

    Vogel, Sven K; Petrasek, Zdenek; Heinemann, Fabian; Schwille, Petra

    2013-01-01

    Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis. DOI: http://dx.doi.org/10.7554/eLife.00116.001 PMID:23326639

  18. Membrane bound O-acyltransferases and their inhibitors.

    PubMed

    Masumoto, Naoko; Lanyon-Hogg, Thomas; Rodgers, Ursula R; Konitsiotis, Antonios D; Magee, Anthony I; Tate, Edward W

    2015-04-01

    Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes. PMID:25849925

  19. Regulatory effects of polyamines on membrane-bound acetylcholinesterase

    PubMed Central

    Kossorotow, A.; Wolf, H. U.; Seiler, N.

    1974-01-01

    The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte `ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na+, K+, Ca2+, Mg2+ and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed. PMID:4462573

  20. Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe

    SciTech Connect

    Middlemas, D.S.; Raftery, M.A.

    1987-03-10

    All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

  1. 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.

  2. Viewing Membrane-Bound Molecular Umbrellas By Parallax Analyses

    PubMed Central

    Kondo, Masaharu; Mehiri, Mohamed

    2010-01-01

    Fluorescence quenching measurements have been made for a series of di-walled and tetra-walled molecular umbrellas having moderate (i.e., hydroxyl-) and strong (i.e., sulfate-) facial hydrophilicity, using Cascade Blue as the fluorophore. Through the use of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine, 1-palmitoyl-2-stearoyl-(5-DOXYL)-sn-glycero-3-phosphocholine, and 1-palmitoyl-2-stearoyl-(12-DOXYL)-sn-glycero-3-phosphocholine as fluorescence quenchers, evidence has been obtained for a membrane-bound state in which the umbrella molecules lie on the surface of the lipid bilayer. In the case of the sulfated molecular umbrellas, evidence has also been obtained for a subpopulation in which the fluorophore lies deeper within the membrane. Probable structures for the shallow-lying and deep-lying molecular umbrellas are discussed. PMID:18783220

  3. A novel membrane-bound glucosyltransferase from Bradyrhizobium japonicum.

    PubMed Central

    Cohen, J L; Miller, K J

    1991-01-01

    Bacteria within the family Rhizobiaceae are distinguished by their ability to infect higher plants. The cell envelope carbohydrates of these bacteria are believed to be involved in the plant infection process. One class of cell envelope carbohydrate, the cyclic beta-1,2-glucans, is synthesized by species within two genera of this family, Agrobacterium and Rhizobium. In contrast, species of the genus Bradyrhizobium, a third genus within this family, appear to lack the capacity for cyclic beta-1,2-glucan biosynthesis. Instead, these bacteria synthesize cyclic glucans containing beta-1,6 and beta-1,3 glycosidic linkages (K.J. Miller, R.S. Gore, R. Johnson, A.J. Benesi, and V.N. Reinhold, J. Bacteriol. 172:136-142, 1990). We now report the initial characterization of a novel membrane-bound glucosyltransferase activity from Bradyrhizobium japonicum USDA 110. Analysis of the product of this glucosyltransferase activity revealed the following: the presence of beta-1,3 and beta-1,6 glycosidic linkages, an average molecular weight of 2,100, and no detectable reducing terminal residues. The glucosyltransferase activity was found to have an apparent Km of 50 microM for for UDP-glucose, and activity was stimulated optimally by Mn2+ ions. On the basis of the structural properties of the in vitro glucan product, it is possible that this membrane-bound glucosyltransferase activity may be responsible for the biosynthesis of cyclic beta-1,6-beta-1,3-glucans by this organism. PMID:1829727

  4. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

    PubMed Central

    Machold, J; Utkin, Y; Kirsch, D; Kaufmann, R; Tsetlin, V; Hucho, F

    1995-01-01

    A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis. Images Fig. 2 PMID:7543679

  5. Co-Immunoprecipitation of Membrane-Bound Receptors

    PubMed Central

    Avila, Julian R.; Lee, Jin Suk; Torii, Keiko U.

    2015-01-01

    The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane. PMID:26097438

  6. 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

  7. Determining the Orientation and Localization of Membrane-Bound Peptides

    PubMed Central

    Hohlweg, Walter; Kosol, Simone; Zangger, Klaus

    2012-01-01

    Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance. PMID:22044140

  8. Frank Deras Jr., Photographer May 1998 CONTEXTUAL VIEW OF TORPEDO ...

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

    Frank Deras Jr., Photographer May 1998 CONTEXTUAL VIEW OF TORPEDO ASSEMBLY BUILDING AND BRIDGE, FACING SOUTHEAST - Torpedo Assembly Building, Eastern end of Yerba Buena Island, San Francisco, San Francisco County, CA

  9. Frank Deras Jr., Photographer June 1998 CONTEXTUAL VIEW OF TORPEDO ...

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

    Frank Deras Jr., Photographer June 1998 CONTEXTUAL VIEW OF TORPEDO ASSEMBLY BUILDING, EAST SIDE, WITH BRIDGE IN BACKGROUND, FACING SOUTHWEST - Torpedo Assembly Building, Eastern end of Yerba Buena Island, San Francisco, San Francisco County, CA

  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. 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.

  12. Inhibition of cation channel function at the nicotinic acethylcholine receptor from Torpedo: Agonist self-inhibition and anesthetic drugs

    SciTech Connect

    Forman, S.A.

    1989-01-01

    Modulation of the nicotinic acethylcholine receptor from Torpedo by cholinergic agonists, local anesthetics, and n-alkanols was studied using {sup 86}Rb{sup +} flux studies in sealed native Torpedo electroplaque membrane vesicles. Reliable concentration-response and kinetic data were obtained using manual ten sec filtration assays in vesicles partially blocked with alpha-bungarotoxin to remove spare receptors and quenched-flow assays to assess initial {sup 86}Rb{sup +} flux rates or the rate of drug-induced receptor inactivation. Concentration response relationships for the agonists acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, and (-)-nicotine are all bell-shape due to stimulation of cation channel opening at low concentrations and inhibition of channels at higher concentrations. The rate of agonist-induced fast desensitization (k{sub d}) increases with (acetylcholine) in parallel with channel activation, suggesting that desensitization proceeds from the open state and/or states in rapid equilibrium with it. At self-inhibitory acetylcholine concentrations, a new rapid inactivation (rate = k{sub f}) is observed before fast desensitization. The rate and extent of rapid inactivation is compatible with bimolecular association between acethylcholine and inhibitory site with K{sub B} = 40 mM.

  13. Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

    PubMed Central

    Eismann, K; Mlejnek, K; Zipprich, D; Hoppert, M; Gerberding, H; Mayer, F

    1995-01-01

    Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm. PMID:7592402

  14. AAA proteases in mitochondria: diverse functions of membrane-bound proteolytic machines.

    PubMed

    Tatsuta, Takashi; Langer, Thomas

    2009-11-01

    FtsH/AAA proteases comprise a distinct family of membrane-bound, ATP-dependent proteases present in eubacteria and eukaryotic cells, where they are confined to mitochondria and chloroplasts. Here, we will summarize versatile functions of AAA proteases within mitochondria, which ensure mitochondrial integrity and cell survival, acting both as quality control and processing enzymes. PMID:19781639

  15. 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.

  16. Membrane-Bound TRAIL Supplements Natural Killer Cell Cytotoxicity Against Neuroblastoma Cells

    PubMed Central

    Sheard, Michael A.; Asgharzadeh, Shahab; Liu, Yin; Lin, Tsen-Yin; Wu, Hong-Wei; Ji, Lingyun; Groshen, Susan; Lee, Dean A.; Seeger, Robert C.

    2013-01-01

    Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of TRAIL (CD253/TNFSF10) due to low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of fourteen neuroblastoma cell lines to the cytotoxicity of NK cells activated with IL-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in fourteen of seventeen (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous IFNγ up-regulated expression of caspase-8 in three of four neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against two of the three lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-IFNγ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells. PMID:23719242

  17. Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR.

    PubMed

    Ullrich, Sandra J; Hellmich, Ute A; Ullrich, Stefan; Glaubitz, Clemens

    2011-05-01

    The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved (31)P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data. PMID:21423170

  18. Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices for Membrane-Bound Enzymes.

    PubMed

    Sun, Wenjie; Vallooran, Jijo J; Fong, Wye-Khay; Mezzenga, Raffaele

    2016-04-21

    Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural characteristics, optical transparency, and capability to coexist with water. This study demonstrates that the in meso immobilized membrane-bound enzyme d-fructose dehydrogenase (FDH) preserves its full activity, follows ideal Michaelis-Menten kinetics, and shows improved stability compared to its behavior in solution. Even after 5 days, the immobilized FDH retained its full activity in meso, whereas a model hydrophilic enzyme, horseradish peroxidase, maintained only 21% of its original activity. We reason that the lipidic bilayers in the three-dimensional structures of cubic mesophases provide an ideal environment for the reconstitution of a membrane-bound enzyme. The preserved activity, long-term stability, and reusability demonstrate that these hybrid nanomaterials are ideal matrices for biosensing and biocatalytic fuel cell applications. PMID:27050734

  19. 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

  20. 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.

  1. 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. PMID:26922180

  2. 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.

  3. 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.

  4. Effect of feeding lipids recovered from fish processing waste by lactic acid fermentation and enzymatic hydrolysis on antioxidant and membrane bound enzymes in rats.

    PubMed

    Rai, Amit Kumar; Bhaskar, N; Baskaran, V

    2015-06-01

    Fish oil recovered from fresh water fish visceral waste (FVW-FO) through lactic acid fermentation (FO-LAF) and enzymatic hydrolysis (FO-EH) were fed to rats to study their influence on lipid peroxidation and activities of antioxidant and membrane bound enzyme in liver, heart and brain. Feeding of FO-LAF and FO-EH resulted in increase (P < 0.05) in lipid peroxides level in serum, liver, brain and heart tissues compared to ground nut oil (control). Activity of catalase (40-235 %) and superoxide dismutase (17-143 %) also increased (P < 0.05) with incremental level of EPA + DHA in diet. The increase was similar to cod liver oil fed rats at same concentration of EPA + DHA. FO-LAF and FO-EH increased (P < 0.05) the Na(+)K(+) ATPase activity in liver and brain microsomes, Ca(+)Mg(+) ATPase in heart microsome and acetylcholine esterase in brain microsomes when fed with 5 % EPA + DHA. There was also significant change in fatty acid composition and cholesterol/phospholipid ratio in microsomes of rat fed with FVW-FO. Feeding FVW-FO recovered by biotechnological approaches enhanced the activity of antioxidant enzymes in tissues, modulates the activities of membrane bound enzymes and improved the fatty acid composition in microsomes of tissues similar to CLO. Utilization of these processing wastes for the production of valuable biofunctional products can reduce the mounting economic values of fish oil and minimize the environmental pollution problems. PMID:26028754

  5. 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

  6. The Membrane-Bound Form of IL-17A Promotes the Growth and Tumorigenicity of Colon Cancer Cells

    PubMed Central

    Van Anh, Do Thi; Park, Sang Min; Lee, Hayyoung; Kim, Young Sang

    2016-01-01

    Interleukin-17A is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene encoding it into CT26 colon cancer cells, either in a secretory or a membrane-bound form. Expression of the membrane-bound form on CT26 cells dramatically enhanced their proliferation in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression: after synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 in the cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membrane-bound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases. PMID:27378226

  7. 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

  8. 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.

  9. A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

    PubMed

    Lonsdale, Richard; Rouse, Sarah L; Sansom, Mark S P; Mulholland, Adrian J

    2014-07-01

    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

  10. 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

  11. 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.

  12. Molecular characterization of structural genes coding for a membrane bound hydrogenase in Methylococcus capsulatus (Bath).

    PubMed

    Csáki, R; Hanczár, T; Bodrossy, L; Murrell, J C; Kovács, K L

    2001-12-18

    The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo. PMID:11750803

  13. Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin

    PubMed Central

    Perlińska, Agata

    2014-01-01

    Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche. PMID:25426338

  14. (/sup 14/C)chloroacetylcholine as an advantageous affinity label of the acetylcholine receptor

    SciTech Connect

    Bodmer, D.M.; Sin-Ren, A.C.; Waser, P.G.

    1987-01-01

    The alkylating agent (/sup 14/C)chloroacetylcholine perchlorate ((/sup 14/C) ClACh) was synthesized and used for affinity labelling of the nicotinic acetylcholine receptor from Torpedo marmorata. Solubilized and affinity-purified receptor proteins were reduced and alkylated according to the bromoacetylcholine-method. Covalent binding of (/sup 14/C) ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the alpha-subunit. Halogen substitution of acetylcholine by chlorine and insertion of a /sup 14/C-isotope instead of the widely used /sup 3/H resulted in favorable properties of the affinity label.

  15. Binding of cationized and native ferritin to cellular structures of the electric organ of Torpedo marmorata.

    PubMed

    Gerbracht, U; Zimmermann, H

    1983-01-01

    The distribution of polycationic and polyanionic binding sites in the electric organ of Torpedo marmorata was investigated by incubation of tissue with native (NF) ferritin. 1) Collagen fibrils from the electric organ carry rosettes of polyanionic sites on their surface with a periodicity of 60 nm, corresponding to the pattern of crossbanding in collagen fibrils. The CF-binding sites are abut 30 nm in size and project 20 nm beyond the surface of the fibril. 2) As revealed by incubation of tissue homogenates, CF heavily stains the intraperiod line of the axonal myelin and also tubular structures in the axonal cytoplasm. 3) Neither the extracellular aspects of the pre- nor the postsynaptic membrane became labeled with either NF or CF. After incubation of tissue homogenates. labeling of the electron-dense material of the cytoplasmic aspect of the postsynaptic membrane was observed with NF and, in particular, with CF. The ventral basal lamina of the electroplaque cell revealed uniform labeling with NF. In contrast, CF-binding sites were distributed in the lamina densa of the basal lamina as a lattice of discrete binding sites, approximately 45 nm in diameter. The presence of polyanionic sites in the basal lamina, which also proceeds through the synaptic cleft, suggests the existence of a diffusion barrier for the released neurotransmitter acetylcholine. It is proposed that this facilitates hydrolysis of acetylcholine in the synaptic cleft and recirculation of the products of hydrolysis to the axon terminal. PMID:6850761

  16. 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. PMID:25576305

  17. Membrane-bound NAC transcription factors in maize and their contribution to the oxidative stress response.

    PubMed

    Wang, Dexin; Yu, Yanchong; Liu, Zhenhua; Li, Shuo; Wang, Zeli; Xiang, Fengning

    2016-09-01

    NAC membrane-bound transcription factors (NTM1-like, NTL proteins) participate in the regulation of plant development and the abiotic stress response. While their function has been thoroughly explored in Arabidopsis thaliana, this is not the case in maize. Seven ZmNTL genes were identified by an in silico scan of relevant genome sequence. All seven included a NAC domain at their N terminus, and an α-helical membrane-bound structure domain in their C terminal region. Based on their gene structure and content of conserved motifs, the seven sequences were distributed into four clades. Six of the seven ZmNTLs were associated with the plasma membrane, and the remaining one with the endoplasmic reticulum. ZmNTL2-7 were more strongly transcribed in the stem than in either the leaf or root, while ZmNTL1 transcript abundance was highest in the leaf. When the plants were exposed to either abscisic acid or hydrogen peroxide treatment, all seven genes were up-regulated in the root and stem and down-regulated in the leaf. The heterologous expression of ZmNTL1-ΔTM, 2-ΔTM and 5-ΔTM in A. thaliana reduced the level of sensitivity of the plant to hydrogen peroxide. PMID:27457981

  18. Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation

    PubMed Central

    Takagi, Shinsuke; Saito, Yoriko; Hijikata, Atsushi; Tanaka, Satoshi; Watanabe, Takashi; Hasegawa, Takanori; Mochizuki, Shinobu; Kunisawa, Jun; Kiyono, Hiroshi; Koseki, Haruhiko; Ohara, Osamu; Saito, Takashi; Taniguchi, Shuichi; Shultz, Leonard D.

    2012-01-01

    In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)–expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45+ cell engraftment and human CD45+CD33+ myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells. PMID:22279057

  19. Next generation SPR technology of membrane-bound proteins for ligand screening and biomarker discovery

    PubMed Central

    Maynard, Jennifer A.; Lindquist, Nathan C.; Sutherland, Jamie N.; Lesuffleur, Antoine; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

    2009-01-01

    Technology based on surface plasmon resonance (SPR) has allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions, from quantitative measurements of binding kinetics and thermodynamics and concentrations in complex samples to epitope analysis. SPR has become the gold standard in industrial and academic settings, in which typically the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, the technology is only beginning to be adapted to the needs of membrane-bound proteins. Including G protein-coupled receptors (GPCR), ion channels and other growth, immune and cellular receptors, these proteins are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcore™, have been adapted for membrane protein analysis by building supported lipid layers or vesicle capture on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss successes with current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of GPCR ligands, biomarker discovery involving membrane bound proteins and basic cellular biology. PMID:19918786

  20. Membrane-bound globin X protects the cell from reactive oxygen species.

    PubMed

    Koch, Jonas; Burmester, Thorsten

    2016-01-01

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. PMID:26631962

  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. 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.

  3. 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. PMID:26104674

  4. The Tumor Necrosis Factor Receptor Stalk Regions Define Responsiveness to Soluble versus Membrane-Bound Ligand

    PubMed Central

    Richter, Christine; Messerschmidt, Sylvia; Holeiter, Gerlinde; Tepperink, Jessica; Osswald, Sylvia; Zappe, Andrea; Branschädel, Marcus; Boschert, Verena; Mann, Derek A.; Scheurich, Peter

    2012-01-01

    The family of tumor necrosis factor receptors (TNFRs) and their ligands form a regulatory signaling network that controls immune responses. Various members of this receptor family respond differently to the soluble and membrane-bound forms of their respective ligands. However, the determining factors and underlying molecular mechanisms of this diversity are not yet understood. Using an established system of chimeric TNFRs and novel ligand variants mimicking the bioactivity of membrane-bound TNF (mTNF), we demonstrate that the membrane-proximal extracellular stalk regions of TNFR1 and TNFR2 are crucial in controlling responsiveness to soluble TNF (sTNF). We show that the stalk region of TNFR2, in contrast to the corresponding part of TNFR1, efficiently inhibits both the receptor's enrichment/clustering in particular cell membrane regions and ligand-independent homotypic receptor preassembly, thereby preventing sTNF-induced, but not mTNF-induced, signaling. Thus, the stalk regions of the two TNFRs not only have implications for additional TNFR family members, but also provide potential targets for therapeutic intervention. PMID:22547679

  5. 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

  6. Arrhenius plot behavior of a. gamma. -radiation-releasable, membrane-bound exonuclease

    SciTech Connect

    Mitchel, R.E.J.

    1981-11-01

    The activation energy of a membrane-bound exonuclease in Micrococcus radiodurans has been measured and the effect of ionizing radiation damage in this sytem explore. The Arrhenius plot for the native bound enzyme was found to be biphasic and the calculated activation energies and transition temperature for the enzymatic reaction were not changed when the enzyme was: (1) solubilized from the membrane with its covalently bound lipid anchor attached, (2) released from the membrane by ionizing radiation, which cleaves off the covalently attached lipid and converts the enzyme from a dimer to a monomer, (3) attached to the membrane after exposure to ionizing radiation under oxic or anoxic conditions, and (4) attached to the membrane in the presence of 10 mM CHCl/sub 3/. Since other membrane-bound enzymes have been shown to be sensitive to membrane perturbations while this one was not, the results suggest that various perturbants, including ionizing radiation, may have differential effects on such enzymes.

  7. Genetic Reconstitution of Functional Acetylcholine Receptor Channels in Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Claudio, Toni; Green, W. N.; Hartman, Deborah S.; Hayden, Deborah; Paulson, Henry L.; Sigworth, F. J.; Sine, Steven M.; Swedlund, Anne

    1987-12-01

    Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

  8. 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

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

    PubMed

    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

  10. 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

  11. 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

  12. The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase

    PubMed Central

    Houslay, Miles D.; Tipton, Keith F.

    1973-01-01

    1. A preparation of a partly purified mitochondrial outer-membrane fraction suitable for kinetic investigations of monoamine oxidase is described. 2. An apparatus suitable for varying the O2 concentration in a spectrophotometer cuvette is described. 3. The reaction catalysed by the membrane-bound enzyme is shown to proceed by a double-displacement (Ping Pong) mechanism, and a formal mechanism is proposed. 4. KCN, NaN3, benzyl cyanide and 4-cyanophenol are shown to be reversible inhibitors of the enzyme. 5. The non-linear reciprocal plot obtained with impure preparations of benzylamine, which is typical of high substrate inhibition, is shown to be due to aldehyde contamination of the substrate. PMID:4778271

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

    PubMed

    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. PMID:27115344

  14. Dynamics of a membrane-bound tryptophan analog in environments of varying hydration: a fluorescence approach.

    PubMed

    Chattopadhyay, Amitabha; Arora, Ajuna; Kelkar, Devaki A

    2005-12-01

    Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w (o)). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration. PMID:16184387

  15. Methods for measuring Class I membrane-bound hyaluronan synthase activity.

    PubMed

    Weigel, Paul H; Padgett-McCue, Amy J; Baggenstoss, Bruce A

    2013-01-01

    Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes. PMID:23765666

  16. 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.

  17. 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

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

    PubMed

    Flanagan, Lindsey A; Parkin, Alison

    2016-02-15

    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

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

    PubMed Central

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

    2016-01-01

    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. PMID:25749018

  1. 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. PMID:27422658

  2. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG

    PubMed Central

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T.

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG’s inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  3. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

    PubMed

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  4. 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

  5. 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

  6. A membrane-bound synthetic receptor that promotes growth of a polymeric coating at the bilayer-water interface.

    PubMed

    Liu, Ying; Young, Michael C; Moshe, Orly; Cheng, Quan; Hooley, Richard J

    2012-07-27

    Primed for action: Atom-transfer radical polymerization (ATRP) can be promoted at a bilayer-water interface by anchoring initiator molecules (see scheme; red) in a membrane-bound synthetic receptor (yellow). The bilayer is formed on a calcinated nanofilm (gray) on a gold surface. PMID:22730162

  7. High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

    PubMed Central

    Sandee, Duanpen

    2011-01-01

    P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins. PMID:21586563

  8. In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

    PubMed Central

    Grubman, Marvin J.; Ehrenfeld, Ellie; Summers, Donald F.

    1974-01-01

    Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P88. In vitro synthesized protein P88 does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes. Images PMID:4368799

  9. CAST FLOOR WITH VIEW OF TORPEDO LADLE (BENEATH CAST FLOOR) ...

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

    CAST FLOOR WITH VIEW OF TORPEDO LADLE (BENEATH CAST FLOOR) AND KEEPERS OF THE CAST HOUSE FLOOR, S.L. KIMBROUGH AND DAVID HOLMES. - U.S. Steel, Fairfield Works, Blast Furnace No. 8, North of Valley Road, West of Ensley-Pleasant Grove Road, Fairfield, Jefferson County, AL

  10. 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

  11. Diversity of Membrane-Bound Nitrate Reductase Genes in Geothermal Springs

    NASA Astrophysics Data System (ADS)

    Poret-Peterson, A. T.; Schwegel, R.; Elser, J. J.; Shock, E.; Anbar, A. D.

    2010-12-01

    Yellowstone National Park (YNP) harbors an array of hot springs with diverse geochemical properties encompassing gradients of pH (<1 to >9), temperature (ambient to boiling), oxygen levels, metal and nutrient concentrations. Such geothermal features provide ideal settings to study nitrogen (N) cycling in high temperature aquatic environments. Our current understanding of N cycle dynamics in hydrothermal systems comes mainly from the study of nitrogen fixation and nitrification. Indeed, research in these areas has extended the upper temperature limits for both processes to above 80°C and stimulated new thoughts on these processes at the cellular and organismal levels. Denitrification at elevated temperatures, on the other hand, has received com-paratively little attention. Here, we use functional gene markers to explore denitrification in YNP hydrothermal springs. During two consecutive summers, we collected sediment and microbial mat samples from various geothermal features for analysis of genes for denitrification and characterization of geochemical parameters (e.g., pH, temperature, relative abundance of trace metals, etc.). Genes encoding putative membrane-bound nitrate reductase (narG)were amplified from sediments and microbial mats of hot springs ranging in temperature from 50°C up to 92°C. Phylogenetic analysis of these genes show that they are most closely related to narG sequences from hyperthermophilic archaea.

  12. Endocytic Trafficking of Membrane-Bound Cargo: A Flotillin Point of View

    PubMed Central

    Meister, Melanie; Tikkanen, Ritva

    2014-01-01

    The ubiquitous and highly conserved flotillin proteins, flotillin-1 and flotillin-2, have been shown to be involved in various cellular processes such as cell adhesion, signal transduction through receptor tyrosine kinases as well as in cellular trafficking pathways. Due to the fact that flotillins are acylated and form hetero-oligomers, they constitutively associate with cholesterol-enriched lipid microdomains. In recent years, such microdomains have been appreciated as platforms that participate in endocytosis and other cellular trafficking steps. This review summarizes the current findings on the role of flotillins in membrane-bound cargo endocytosis and endosomal trafficking events. We will discuss the proposed function of flotillins in endocytosis in the light of recent findings that point towards a role for flotillins in a step that precedes the actual endocytic uptake of cargo molecules. Recent findings have also revealed that flotillins may be important for endosomal sorting and recycling of specific cargo molecules. In addition to these aspects, the cellular trafficking pathway of flotillins themselves as potential cargo in the context of growth factor signaling will be discussed. PMID:25019426

  13. 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

  14. C. elegans uses Liquid-Liquid Demixing for the Assembly of Non-Membrane-Bound Compartments

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Juelicher, Frank; Diaz Delgadillo, Andres Felipe; Jawerth, Louise; Hyman, Anthony A.; Department Biological Physics Team; Hyman Lab Collaboration

    2015-03-01

    P granules are liquid cytoplasmic RNA/Protein condensates known to determine the germ lineage in Caenorhabditis elegans. They resemble striking similarities with liquid droplets, such as dripping, shearing and wetting. Assuming that P granules are liquid-like we consider how they form in the crowded cytoplasm. Using confocal and light-sheet microscopy, P granule formation in-vivo and in-vitro is shown to share all hallmarks with a liquid-liquid phase-separation. Specifically, demixing is determined by temperature and concentration, the droplet formation is reversible with respect to temperature quenches and there is evidence for droplet growth due to coalescence and Ostwald-ripening. Liquid-liquid demixing in-vivo breaks the paradigmatic view that a molecular machinery is necessary to build up organelles through complex biological pathways. Instead we propose that P granules form following a Flory-Huggins model. Liquid-liquid demixing could also serve as a mechanism for the assembly of non-membrane-bound compartments in other living organisms.

  15. 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

  16. Efficient photoinduced orthogonal energy and electron transfer reactions via phospholipid membrane-bound donors and acceptors

    SciTech Connect

    Clapp, P.J.; Armitage, B.; Roosa, P.; O'Brien, D.F. )

    1994-10-05

    A three component, liposome-bound photochemical molecular device (PMD) consisting of energy and electron transfer reactions is described. Bilayer membrane surface-associated dyes, 5,10,15,20-tetrakis[4-(trimethylammonio)-phenyl]-21H,2 3H-porphine tetra-p-tosylate salt and N,N[prime]-bis[(3-trimethylammonio)propyl]thiadicarbocya nine tribromide, are the energy donor and acceptor, respectively, in a blue light stimulated energy transfer reaction along the vesicle surface. The electronically excited cyanine is quenched by electron transfer from the phospholipid membrane bound triphenylbenzyl borate anion, which is located in the lipid bilayer interior. The PMD exhibits sequential reactions following electronic excitation with the novel feature that the steps proceed with orthogonal orientation: energy transfer occurs parallel to the membrane surface, and electron transfer occurs perpendicular to the surface. Photobleaching and fluorescence quenching experiments verify the transfer reactions, and Stern-Volmer analysis was used to estimate the reaction rate constants. At the highest concentrations examined of energy and electron acceptor ca. 60% of the photoexcited porphyrins were quenched by energy transfer to the cyanine. 56 refs., 6 figs., 3 tabs.

  17. 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.

  18. 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. PMID:25863612

  19. Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    PubMed

    Chesterman, Chelsy; Jia, Zongchao

    2016-09-01

    Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. PMID:26363120

  20. [Purification and properties of membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

    PubMed

    Gvozdev, R I; Tukhvatullin, I A; Tumanova, L V

    2008-01-01

    Membrane fraction of Methylococcus capsulatus (strain M) were treated with [14C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (alpha), 27 (beta), and 25 (gamma) kDa in the ratio 1:1:1. The radiolabel was contained in the beta-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2-4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed. PMID:18946992

  1. Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.

    PubMed Central

    Dailey, H A

    1977-01-01

    The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme. Images PMID:21163

  2. Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins.

    PubMed

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

    2016-09-01

    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

  3. Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.

    PubMed

    Luoto, Heidi H; Nordbo, Erika; Malinen, Anssi M; Baykov, Alexander A; Lahti, Reijo

    2015-04-15

    Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases. PMID:25662511

  4. Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes.

    PubMed

    Fonfría, Eva S; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Otero, Paz; Espiña, Begoña; Louzao, M Carmen; Alvarez, Mercedes; Botana, Luis M

    2010-08-01

    Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with alpha-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 +/- 1.9%. The quantification range for this toxin in mussel was 40-200 microg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay. PMID:20382103

  5. Agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action

    SciTech Connect

    Forman, S.A.; Firestone, L.L.; Miller, K.W.

    1987-05-19

    Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

  6. Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase.

    PubMed

    Silva, Virgília S; Nunes, M Alexandra; Cordeiro, J Miguel; Calejo, Ana I; Santos, Sofia; Neves, Paulo; Sykes, António; Morgado, Fernando; Dunant, Yves; Gonçalves, Paula P

    2007-07-17

    Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3

  7. Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung.

    PubMed Central

    Lizcano, J M; Tipton, K F; Unzeta, M

    1998-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine. PMID:9512463

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

    PubMed

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

    2015-01-01

    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

  9. Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

    PubMed

    Rudenko, Natalia N; Ignatova, Lyudmila K; Ivanov, Boris N

    2007-01-01

    Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids. PMID:17347907

  10. 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

  11. Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

    PubMed

    Lalla-Maharajh, W V; Hall, D O; Cammack, R; Rao, K K; Le Gall, J

    1983-02-01

    The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5. PMID:6303306

  12. Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

    PubMed

    Croyle, Kaitlin; Gibbons, Paul; Light, Christine; Goode, Eric; Durrant, Barbara; Jensen, Thomas

    2016-03-01

    Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification. Zoo Biol. 35:95-103, 2016. © 2016 Wiley Periodicals, Inc. PMID:26890048

  13. [The binuclear iron site of the membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

    PubMed

    Tumanova, L V; Tukhvatullin, I A; Burbaev, T Sh; Gvozdev, R I; Andersson, K K

    2008-01-01

    The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: 47 (alpha), 27 (beta), and 25 kDa (gamma). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4 degrees C exhibited, along with the copper signal of type I with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00. After incubation at -30 degrees C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at -30 degrees C. It was established that this intermediate is a high-spin dimer [Fe(IlI)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(lII)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the beta-subunit and cannot be detected by ESR. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru. PMID:18522275

  14. Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation

    PubMed Central

    2013-01-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme. PMID:23697766

  15. Exploring homo-FRET to quantify the oligomer stoichiometry of membrane-bound proteins involved in a cooperative partition equilibrium.

    PubMed

    Melo, Ana M; Fedorov, Aleksander; Prieto, Manuel; Coutinho, Ana

    2014-09-14

    The establishment of protein-protein interactions between membrane-bound proteins is associated with several biological functions and dysfunctions. Here, an analytical framework that uses energy homo transfer to directly probe quantitatively the oligomerization state of membrane-bound proteins engaged in a three-state cooperative partition is presented. Briefly, this model assumes that monomeric protein molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits. A general equation relating the overall steady-state fluorescence anisotropy of the sample to its fractional labeling was derived by considering explicitly that the anisotropy of mixed oligomers containing i-labeled monomers is inversely proportional to the number of labeled subunits per oligomer (Runnels and Scarlata limit). This method was very robust in describing the electrostatic interaction of Alexa Fluor 488 fluorescently labeled lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the system reaching a high membrane surface density of the protein (at a low lipid-to-protein (L/P) molar ratio). The occurrence of energy homo transfer-induced fluorescence depolarization was further confirmed by measuring the anisotropy decays of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data obtained under a wide range of experimental conditions (variable anionic lipid content of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a stoichiometry of k = 6 ± 1. This study illustrates that even in the presence of a coupled partition-oligomerization equilibrium, steady-state anisotropy measurements provide a simple and reliable tool to monitor the self-assembly of membrane-bound proteins. PMID:24722583

  16. Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound.

    PubMed

    Nishikiori, Masaki; Dohi, Koji; Mori, Masashi; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

    2006-09-01

    Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity. PMID:16912296

  17. 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. PMID:27138824

  18. 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. PMID:26481477

  19. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    PubMed

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. PMID:26906096

  20. ESTRADIOL-INDUCED ENHANCEMENT OF OBJECT MEMORY CONSOLIDATION INVOLVES HIPPOCAMPAL ERK ACTIVATION AND MEMBRANE-BOUND ESTROGEN RECEPTORS

    PubMed Central

    Fernandez, Stephanie M.; Lewis, Michael C.; Pechenino, Angela S.; Harburger, Lauren L.; Orr, Patrick T.; Gresack, Jodi E.; Schafe, Glenn E.; Frick, Karyn M.

    2009-01-01

    The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen, 17β-estradiol (E2). Here, we asked whether E2 modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal (i.p.) injection of 0.2 mg/kg E2 significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 hour after injection. Second, we show that E2 administered i.p. (0.2 mg/kg) or via intrahippocampal infusion (5.0 μg/side) immediately after training in an object recognition task significantly enhances memory retention, and that the beneficial effect of i.p. E2 is blocked by dorsal hippocampal inhibition of ERK activation. Third, using bovine serum albumin-conjugated 17β-estradiol (BSA-E2), we demonstrate that E2 binding at membrane-bound ERs can increase dorsal hippocampal ERK activation and enhance object memory consolidation in an ERK-dependent manner. Fourth, we show that this effect is independent of nuclear ERs, but is dependent on the dorsal hippocampus. By demonstrating that E2 enhances memory consolidation via dorsal hippocampal ERK activation, this study is the first to identify a specific molecular pathway by which E2 modulates memory and to demonstrate a novel role for membrane-bound ERs in mediating E2-induced improvements in hippocampal memory consolidation. PMID:18753366

  1. Naval Underwater Systems Center torpedo data acquisition system design update

    SciTech Connect

    Adams, D.J.; Beshears, D.L.; Capps, G.J.; Jordan, J.K.; Napier, R.W.; Ott, G.W.; Scudiere, M.B.; White, C.P.; Nodine, R.N.; Todd, R.A.

    1988-10-01

    In the process of developing technology for torpedo applications, the Naval Underwater Systems Center test vehicles are instrumented with sensors (and signal processors), and their outputs are recorded during in water exercises. This paper defines the design for both the wet'' and dry'' systems. The wet system will be the on-board torpedo equipment used to monitor and record a multitude of transducer of transducer signals. The dry system will be used for programming the wet system, handling the test data, and performing quick-look signal analyses. The system will be used to monitor, record, and analyze an enormous volume of data from transducer signals during either routine or development in-water exercises. 24 figs.

  2. 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

  3. Structure and superorganization of acetylcholine receptor–rapsyn complexes

    PubMed Central

    Zuber, Benoît; Unwin, Nigel

    2013-01-01

    The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor–rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters. PMID:23754381

  4. 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

  5. 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

  6. 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

  7. In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes.

    PubMed

    Fraser, P D; Sandmann, G

    1992-05-29

    In vitro assays have been developed for three membrane-bound carotenogenic enzymes, phytoene desaturase, lycopene cyclase and beta-carotene hydroxylase, expressed in Escherichia coli. Transformants of E. coli containing different deletion constructs of the Erwinia herbicola carotenogenic gene cluster were employed, allowing the estimation of enzyme activities without interference from subsequent reactions. New HPLC systems were developed to separate substrates and reaction products enabling the determination of radioactivity on-line. The newly developed assays facilitate the purification of these enzymes which have never been isolated before. PMID:1599492

  8. 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

  9. Cholinergic ligand interactions with acetylcholine receptor proteins and solvent interactions with N,N-dialkylnicotinamides

    SciTech Connect

    Bean, J.W.

    1987-01-01

    A dual-chambered flow dialysis nuclear counting apparatus was used to monitor cholinergic ligand induced displacement of {sup 155}Eu{sup 3+} from acetylcholine receptor proteins. Acetylcholine, nicotine and carbamylcholine induced similar rates of displacement of {sup 155}Eu{sup 3+} probes of calcium binding sites in receptor proteins from wild type Drosophila melanogaster and Torpedo californica. The receptor isolated from a nicotine resistant strain of Drosophila melanogaster displayed an altered dependency of cholinergic ligand induced cation displacement with respect to the other two receptor proteins. Both Drosophila strains' solubilized receptor proteins migrated as three bands of molecular weights 68,000, 66,000, and 60,000 on denaturing polyacrylamide gels. Carbon-13 NMR techniques were employed to examine the effects of solvent environment on rotational energy barriers in a series of molecules related to the analeptic, nikethamide: N,N-dimethylnicotinamide, 1-nicotinoyl piperidine, and N,N-dipropylnicotinamide.

  10. 6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological evaluation on nicotinic acetylcholine receptors.

    PubMed

    Duroure, Leslie; Jousseaume, Thierry; Aráoz, Rómulo; Barré, Elvina; Retailleau, Pascal; Chabaud, Laurent; Molgó, Jordi; Guillou, Catherine

    2011-12-01

    Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched mixtures were tested for their biological activities on Xenopus oocytes either expressing (human α4β2) or having incorporated (Torpedoα1(2)βγδ) nicotinic acetylcholine receptors (nAChRs). These spiroimine analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins. PMID:22024965

  11. 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

  12. 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. PMID:26341797

  13. Oligomerization of Membrane-Bound Diphtheria Toxin (CRM197) Facilitates a Transition to the Open Form and Deep Insertion

    PubMed Central

    Kent, M. S.; Yim, H.; Murton, J. K.; Satija, S.; Majewski, J.; Kuzmenko, I.

    2008-01-01

    Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane. PMID:18055530

  14. 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

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

    PubMed

    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

  17. Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

    PubMed Central

    Raja, S M; Rawat, S S; Chattopadhyay, A; Lala, A K

    1999-01-01

    The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin. PMID:10049328

  18. (-)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

    PubMed

    Arias, Hugo R; Ortells, Marcelo O; Feuerbach, Dominik

    2013-11-01

    The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca(2+) influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48 μM, respectively, (b) binds to the [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6' and 13' in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9' is the minimum structural component for (-)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. PMID:23917086

  19. 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

  20. 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

  1. 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.

  2. 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

    2016-01-01

    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

  3. Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH:ubiquinone oxidoreductase (complex I)

    PubMed Central

    Gostimskaya, Irina S.; Grivennikova, Vera G.; Cecchini, Gary; Vinogradov, Andrei D.

    2008-01-01

    Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with Kd (~10−8 M at pH 10.0). PMID:18037377

  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. PMID:26152359

  5. Quantum Chemical Calculations of Amide-15N Chemical Shift Anisotropy Tensors for a Membrane-Bound Cytochrome b5

    PubMed Central

    Pandey, Manoj Kumar; Ramamoorthy, Ayyalusamy

    2013-01-01

    There is considerable interest in determining amide-15N chemical shift anisotropy (CSA) tensors from biomolecules and understanding their variation for structural and dynamics studies using solution and solid-state NMR spectroscopy and also by quantum chemical calculations. Due to the difficulties associated with the measurement of CSA tensors from membrane proteins, NMR-based structural studies heavily relied on the CSA tensors determined from model systems, typically single crystals of model peptides. In the present study, the principal components of backbone amide-15N CSA tensor have been determined using density functional theory for a 16.7-kDa membrane-bound paramagnetic heme containing protein, cytochrome b5 (cytb5). All the calculations were performed by taking residues within 5Å distance from the backbone amide-15N nucleus of interest. The calculated amide-15N CSA spans agree less well with our solution NMR data determined for an effective internuclear distance rN-H = 1.023 Å and a constant angle β = 18° that the least shielded component (δ11) makes with the N-H bond. The variation of amide-15N CSA span obtained using quantum chemical calculations is found to be smaller than that obtained from solution NMR measurements, whereas the trends of the variations are found to be in close agreement. We believe that the results reported in this study will be useful in studying the structure and dynamics of membrane proteins and heme-containing proteins, and also membrane-bound protein-protein complexes such as cytochromes-b5-P450. PMID:23268659

  6. Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    McTernan, Patrick M; Chandrayan, Sanjeev K; Wu, Chang-Hao; Vaccaro, Brian J; Lancaster, W Andrew; Yang, Qingyuan; Fu, Dax; Hura, Greg L; Tainer, John A; Adams, Michael W W

    2014-07-11

    The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions. PMID:24860091

  7. A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*

    PubMed Central

    Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stéphanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

  8. Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

    PubMed Central

    Poulter, L; Earnest, J P; Stroud, R M; Burlingame, A L

    1989-01-01

    Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide. Images PMID:2771948

  9. Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Shenkarev, Zakhar O; Kasheverov, Igor E; Chugunov, Anton O; Kulbatskii, Dmitrii S; Myshkin, Mikhail Yu; Utkin, Yuri N; Efremov, Roman G; Tsetlin, Victor I; Arseniev, Alexander S; Kirpichnikov, Mikhail P; Dolgikh, Dmitry A

    2016-09-01

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs. PMID:27343701

  10. Dimeric structure of single chloride channels from Torpedo electroplax.

    PubMed Central

    Miller, C; White, M M

    1984-01-01

    The inhibition by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) of Cl- channels from Torpedo electroplax incorporated in planar phospholipid bilayer membranes is studied. DIDS irreversibly and rapidly inhibits the macroscopic conductance of membranes containing many channels. At the single-channel level, the effect of DIDS is more complicated. The uninhibited single channel displays three "substates" of conductances 20, 10, and 0 pS. Short exposure (5-30 s) to 10 microM DIDS converts this three-level active channel into a "conventional" channel of 10-pS conductance. Longer exposure eliminates all channel fluctuations. The results are taken as strong evidence that the Cl- channel is constructed as a functional dimer of identical protein subunits. PMID:6326143

  11. 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.

  12. Permeability control by cholinergic receptors in Torpedo postsynaptic membranes: agonist dose-response relations measured at second and millisecond times.

    PubMed

    Neubig, R R; Cohen, J B

    1980-06-10

    A quantitative analysis of nicotine acetylcholine receptor function in Torpedo postsynaptic membranes is presented. 22Na+ efflux induced by carbamylcholine (Carb) and the partial agonist phenyltrimethylammonium (PTA) is assessed by determining dose-response relations using three approaches: (1) a filtration assay measuring responses on the 10-s time scale, (2) the same filtration assay after blocking different fractions of the receptor sites with alpha-bungarotoxin (alpha-BgTx), and (3) a rapid-mix quenched-flow technique which permits measurement of the initial rate of 22Na+ efflux on the millisecond time scale. The concentrations of agonist producing half-maximal responses in these three assays at 4 degrees C are 13, 150, and 600 microM, respectively, for Carb and 50, 50, and 200 microM, respectively, for PTA. The rate constants for 22Na+ efflux are 1.3 x 10(-4) s-1 in the absence of agonst and 65 s-1 and 0.8 s-1 in the presence of maximal concentrations of Carb and PTA, respectively, representing a stimulation of 5 x 10(5) by Carb. The Hill coefficient for the Carb response, expressed as rate constants for 22N+ efflux, is 1.97 +/- 0.06 for Carb concentrations between 3 microM and 1 mM. The inhibition of the agonist-stimulated 22Na+ efflux by alpha-BgTx is compatible with two alpha-BgTx (and acetylcholine) sites per functional unit. Inhibition of Carb responses (slow assay) by d-tubocurarine appears competitive with a KI approximately 0.5 microM, while responses to PTA are inhibited noncompetitively with KI = 0.3 microM. This paradox is due to the presence of spare receptors and to complexities in the binding of dTC to the nicotinic acetylcholine receptor. Determination of responses without the complication of spare receptors allows a meaningful comparison to direct measurements of agonist and antagonist binding in the same system. A model is proposed to account for both binding and response. PMID:7397104

  13. 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. PMID:25894196

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

    PubMed Central

    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. PMID:25894196

  15. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  16. 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

  17. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae.

    PubMed

    Dalia, Ankur B; Lazinski, David W; Camilli, Andrew

    2014-01-01

    Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named "TfoS" (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. IMPORTANCE Naturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the family Vibrionaceae become naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural

  18. Counting Bungarotoxin Binding Sites of Nicotinic Acetylcholine Receptors in Mammalian Cells with High Signal/Noise Ratios

    PubMed Central

    Simonson, Paul D.; DeBerg, Hannah A.; Ge, Pinghua; Alexander, John K.; Jeyifous, Okunola; Green, William N.; Selvin, Paul R.

    2010-01-01

    Nicotinic acetylcholine receptors are some of the most studied synaptic proteins; however, many questions remain that can only be answered using single molecule approaches. Here we report our results from single α7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophore-labeled bungarotoxin, we can image individual receptors and count the number of bungarotoxin-binding sites in receptors expressed in HEK 293 cells. Our results indicate that there are two bungarotoxin-binding sites in neuromuscular junction receptors, as expected, and five in α7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound proteins at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques. PMID:21081055

  19. Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro.

    PubMed Central

    Hall, C; Lim, L

    1981-01-01

    Free and membrane-bound polyribosomes were isolated from the rat forebrain during its development. Polyadenylated RNA [poly(A)+ RNA] was isolated from both fractions, by using oligo(dT)-cellulose chromatography, and its composition studied by translating the poly(A)+ RNA in vitro in reticulocyte lysates. Electrophoretic analysis of the translation products showed that both free and membrane-bound polyribosomal poly(A)+ RNA gave many common components, but that there were also distinct differences in the protein composition of the products of the two fractions. Several proteins, of mol.wts. 39 000, 37 000, 31 000, 27 000 and 17 000, appeared to be products predominantly of free polyribosomal poly(A)+ RNA, whereas others, of mol.wt. 47 000, 33 000, 24 000 and 21 000 were specific to the membrane-bound polyribosomal poly(A)+ RNA fraction. More developmental changes were observed in the translational products of the membrane-bound poly(A)+ RNA fraction. Proteins of mol.wts. 33 000 and 21 000, which were predominant components of the translational products of this fraction when isolated from 10-day and older rats, were not present in translational products derived from preparations isolated from 3-day-old rats. The developmental appearance of these proteins as translational products of the membrane-bound poly(A)+ RNA suggests the appearance of new mRNA species. These transcriptional changes are discussed in relation to processes involved in brain differentiation, including myelination. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:6171267

  20. 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. PMID:26656912

  1. 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

  2. A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4

    PubMed Central

    Hu, Xiao-Min; Xu, Yan-Rui; Yan, Ru; Sun, Shu-Liang; Dong, Hong-Liang; Wang, Jun; Gao, Xiao-Ming

    2015-01-01

    Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease. PMID:26649297

  3. 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. PMID:27382350

  4. Nanoscale patterning of membrane-bound proteins formed through curvature-induced partitioning of phase-specific receptor lipids.

    PubMed

    Ogunyankin, Maria O; Huber, Dale L; Sasaki, Darryl Y; Longo, Marjorie L

    2013-05-21

    This work describes a technique for forming high-density arrays and patterns of membrane-bound proteins through binding to a curvature-organized compositional pattern of metal-chelating lipids (Cu(2+)-DOIDA or Cu(2+)-DSIDA). In this bottom-up approach, the underlying support is an e-beam formed, square lattice pattern of hemispheres. This curvature pattern sorts Cu(2+)-DOIDA to the 200 nm hemispherical lattice sites of a 600 nm × 600 nm unit cell in Ld - Lo phase separated lipid multibilayers. Binding of histidine-tagged green fluorescent protein (His-GFP) creates a high density array of His-GFP-bound pixels localized to the square lattice sites. In comparison, the negative pixel pattern is created by sorting Cu(2+)-DSIDA in Ld - Lβ' phase separated lipid multibilayers to the flat grid between the lattice sites followed by binding to His-GFP. Lattice defects in the His-GFP pattern lead to interesting features such as pattern circularity. We also observe defect-free arrays of His-GFP that demonstrate perfect arrays can be formed by this method suggesting the possibility of using this approach for the localization of various active molecules to form protein, DNA, or optically active molecular arrays. PMID:23642033

  5. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  6. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making.

    PubMed

    Tuladhar, Rubina; Lum, Lawrence

    2015-04-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  7. 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

  8. 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. PMID:27271742

  9. 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

  10. 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.

  11. Identification of a Membrane-Bound Transcriptional Regulator That Links Chitin and Natural Competence in Vibrio cholerae

    PubMed Central

    Dalia, Ankur B.; Lazinski, David W.; Camilli, Andrew

    2014-01-01

    ABSTRACT Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named “TfoS” (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. PMID:24473132

  12. Neonatal transfer of membrane-bound stem cell factor improves survival and heart function in aged mice after myocardial ischemia.

    PubMed

    Sun, Zhuo; Lee, Chyan-Jang; Mejia-Guerrero, Salvador; Zhang, Yuemei; Higuchi, Koji; Li, Ren-Ke; Medin, Jeffrey A

    2012-12-01

    Stem cell mobilization to injured tissue contributes to neovascularization, resulting in regeneration after myocardial infarction (MI). We previously showed that direct cardiac injection of a recombinant lentivirus (LV) that engineers expression of membrane-bound stem cell factor (mSCF) improves outcomes immediately after MI. In this study, we evaluated the effect of neonatal LV/mSCF transduction on MI outcomes in aged mice. We constructed a recombinant LV harboring an α-myosin heavy chain promoter that drives mSCF expression and injected it into the temporal vein of neonatal mice. One year later, sustained expression of mSCF in the adult mouse hearts was detected by genomic and quantitative RT-PCR and immunohistochemistry. To evaluate the contribution of neonatal LV/mSCF delivery to recovery from MI, we induced an MI in adult LV/mSCF-transduced, LV only-transduced, and nontransduced control mice. Strikingly, LV/mSCF transduction reduced infarct scar size, enhanced angiogenesis, improved ventricular function, and significantly increased survival of the mice. Regional overexpression of CD11b, a marker of monocytes and proangiogenic cells, was observed on monocytes isolated from the infarcted hearts of LV/mSCF-transduced mice. Our data suggest a model of neonatal gene delivery that leads to sustained mSCF expression during adulthood to aid recovery from MI and prevent heart failure. PMID:22998370

  13. 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

  14. 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

  15. 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. PMID:24573306

  16. 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.

  17. 33 CFR 334.1190 - Hood Canal and Dabob Bay, Wash.; naval non-explosive torpedo testing area.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 3 2010-07-01 2010-07-01 false Hood Canal and Dabob Bay, Wash.; 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)...

  18. 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

  19. A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.

    PubMed

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J; Wang, Guodong

    2015-03-01

    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. Top-Down Mass Spectrometry Analysis of Membrane-Bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides.

    PubMed

    Lu, Yue; Zhang, Hao; Cui, Weidong; Saer, Rafael; Liu, Haijun; Gross, Michael L; Blankenship, Robert E

    2015-12-15

    We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 β polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the β polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid. PMID:26574182

  1. 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. PMID:27305962

  2. 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

  3. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor.

    PubMed

    Pérez, Claudia; Fernandez, Luisa E; Sun, Jianguang; Folch, Jorge Luis; Gill, Sarjeet S; Soberón, Mario; Bravo, Alejandra

    2005-12-20

    Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. PMID:16339907

  4. Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection

    PubMed Central

    Verchot, Jeanmarie

    2012-01-01

    Cellular chaperones and folding enzymes play central roles in the formation of positive-strand and negative-strand RNA virus infection. This article examines the key cellular chaperones and discusses evidence that these factors are diverted from their cellular functions to play alternative roles in virus infection. For most chaperones discussed, their primary role in the cell is to ensure protein quality control. They are system components that drive substrate protein folding, complex assembly or disaggregation. Their activities often depend upon co-chaperones and ATP hydrolysis. During plant virus infection, Hsp70 and Hsp90 proteins play central roles in the formation of membrane-bound replication complexes for certain members of the tombusvirus, tobamovirus, potyvirus, dianthovirus, potexvirus, and carmovirus genus. There are several co-chaperones, including Yjd1, RME-8, and Hsp40 that associate with the bromovirus replication complex, pomovirus TGB2, and tospovirus Nsm movement proteins. There are also examples of plant viruses that rely on chaperone systems in the endoplasmic reticulum (ER) to support cell-to-cell movement. TMV relies on calreticulin to promote virus intercellular transport. Calreticulin also resides in the plasmodesmata and plays a role in calcium sequestration as well as glycoprotein folding. The pomovirus TGB2 interacts with RME-8 in the endosome. The potexvirus TGB3 protein stimulates expression of ER resident chaperones via the bZIP60 transcription factor. Up-regulating factors involved in protein folding may be essential to handling the load of viral proteins translated along the ER. In addition, TGB3 stimulates SKP1 which is a co-factor in proteasomal degradation of cellular proteins. Such chaperones and co-factors are potential targets for antiviral defense. PMID:23230447

  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. PMID:22278771

  6. Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

    PubMed

    Ferroni, Felix M; Rivas, María G; Rizzi, Alberto C; Lucca, María E; Perotti, Nora I; Brondino, Carlos D

    2011-10-01

    The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI. PMID:21432624

  7. Catharanthine alkaloids are noncompetitive antagonists of muscle-type nicotinic acetylcholine receptors.

    PubMed

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2010-09-01

    We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (+/-)-epibatidine-induced Ca(2+) influx in TE671-halpha1beta1gammadelta cells with similar potencies (IC(50)=17-25microM), (b) inhibit [(3)H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [(3)H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [(3)H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6'), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20'), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel. PMID:20493225

  8. Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states.

    PubMed

    Arias, Hugo R; Rosenberg, Avraham; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Maciejewski, Ryszard; Jozwiak, Krzysztof; Moaddel, Ruin; Glick, Stanley D; Wainer, Irving W

    2010-06-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization. PMID:20303928

  9. Light-induced reactivation of O2-tolerant membrane-bound [Ni-Fe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus under turnover conditions.

    PubMed

    Ciaccafava, Alexandre; Hamon, Cyrille; Infossi, Pascale; Marchi, Valérie; Giudici-Orticoni, Marie-Thérèse; Lojou, Elisabeth

    2013-10-21

    We report the effect of UV-Vis light on the membrane-bound [Ni-Fe] hydrogenase from Aquifex aeolicus under turnover conditions. Using electrochemistry, we show a potential dependent light sensitivity and propose that a light-induced structural change of the [Ni-Fe] active site is related to an enhanced reactivation of the hydrogenase under illumination at high potentials. PMID:23999766

  10. Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria.

    PubMed

    Adachi, Osao; Hours, Roque A; Shinagawa, Emiko; Akakabe, Yoshihiko; Yakushi, Toshiharu; Matsushita, Kazunobu

    2011-01-01

    In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes. PMID:21897028

  11. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  12. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  13. 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

  14. Fractional vesamicol receptor occupancy and acetylcholine active transport inhibition in synaptic vesicles.

    PubMed

    Kaufman, R; Rogers, G A; Fehlmann, C; Parsons, S M

    1989-09-01

    Vesamicol [(-)-(trans)-2-(4-phenylpiperidino)cyclohexanol] receptor binding and inhibition of acetylcholine (AcCh) active transport by cholinergic synaptic vesicles that were isolated from Torpedo electric organ were studied for 23 vesamicol enantiomers, analogues, and other drugs. Use of trace [3H]vesamicol and [14C]AcCh allowed simultaneous determination of the concentrations of enantiomer, analogue, or drug required to half-saturate the vesamicol receptor (Ki) and to half-inhibit transport (IC50), respectively. Throughout a wide range of potencies for different compounds, the Ki/IC50 ratios varied from 1.5 to 24. Compounds representative of the diverse structures studied, namely deoxyvesamicol, chloroquine, and levorphanol, were competitive inhibitors of vesamicol binding. It is concluded that many drugs can bind to the vesamicol receptor and binding to only a small fraction of the receptors can result in AcCh active transport inhibition. Possible mechanisms for this effect are discussed. PMID:2550778

  15. Two partially unfolded states of Torpedo californica acetylcholinesterase.

    PubMed

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

    1996-09-01

    Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on

  16. Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors

    PubMed Central

    Sennikov, Sergey V.; Vasilyev, Filipp F.; Lopatnikova, Julia A.; Shkaruba, Nadezhda S.; Silkov, Alexander N.

    2014-01-01

    The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFα receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP −609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP −1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP −1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP −3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI −609GT + TNFRII −3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFα receptors and TNFα-mediated signaling. PMID:24782596

  17. Partial proteolysis as a probe of the conformation of the gamma subunit in activated soluble and membrane-bound chloroplast coupling factor 1.

    PubMed

    Schumann, J; Richter, M L; McCarty, R E

    1985-09-25

    Treatments that enhance the latent ATPase activity of the chloroplast coupling factor (CF1) also induce hypersensitivity of the gamma subunit toward trypsin. A number of different gamma subunit cleavage products are formed (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). We have compared the gamma cleavage products of membrane-bound and isolated CF1, activated either by reduction of the gamma disulfide bond or by removal of the epsilon subunit. The gamma subunit of isolated CF1 lacking the epsilon subunit was cleaved to a 27,000-Da species. The same cleavage site became exposed following energy-dependent conformational changes in the membrane-bound enzyme. Activation by reduction of the gamma disulfide bond also exposed this site. However, the gamma subunit of reduced CF1 was cleaved rapidly at an additional site and trypsin treatment gave rise to a 25,000-Da gamma species. The small peptide generated by the second cleavage contains one of the cysteinyl residues of the reduced disulfide bridge of gamma. This peptide dissociates from the enzyme and can be isolated by gel filtration. The close proximity of the trypsin cleavage sites to the disulfide bond of gamma is discussed with respect to the effects of tryptic cleavage on the ATPase activity of CF1. The data indicate that structural changes in a limited region of the gamma subunit strongly influence the catalytic properties of both soluble and membrane-bound CF1. PMID:2864336

  18. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity.

    PubMed Central

    Rajas, F; Rousset, B

    1993-01-01

    We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme. Images Figure 1 Figure 3

  19. Sensing acetylcholine and anticholinesterase compounds.

    PubMed

    Schena, Alberto; Johnsson, Kai

    2014-01-27

    Acetylcholine is a key neurotransmitter, and anticholinesterase agents are essential compounds used as medical drugs, pesticides, and chemical warfare agents. A semisynthetic fluorescence-based probe for the direct, real-time detection of acetylcholine and anticholinesterase compounds is introduced. The probe possesses good sensitivity, tunable detection range, and can be selectively targeted to cell surfaces, thereby making it an attractive tool for applications in analytical chemistry and quantitative biology. PMID:24339043

  20. 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.

  1. Hyperpigmented Torpedo Maculopathy with Pseudo-Lacuna: A 5-Year Follow-Up

    PubMed Central

    Rohl, Austin; Vance, Sushma

    2016-01-01

    Purpose The aim of the study was to describe a case of globally hyperpigmented torpedo maculopathy that also contained a novel central lesion resembling a ‘pseudo-lacuna’. We compare the morphology of the lesion after 5 years of follow-up. Case Presentation An asymptomatic 10-year-old Caucasian male was referred by his optometrist after having found a hyperpigmented lesion on routine dilated examination in 2010. Color fundus photography OS from October 2015 showed a 1.74 × 0.67 mm hyperpigmented oval-shaped lesion temporal to the macula. Since June 2010, the hyperpigmented torpedo lesion appeared to have assumed a more ovoid shape and increased in size in the vertical axis. Centrally, there was a small pearlescent-colored pseudo-lacuna lesion that seemed to also have significantly increased in size since June 2010. Enhanced depth imaging optical coherence tomography of this pseudo-lacuna showed retinal pigment epithelium clumping and migration. Fundus autofluorescence revealed reduced autofluorescence of the torpedo lesion and marked hyperautofluorescence of the pseudo-lacuna. Fluorescein angiography shows no neovascular disease or leakage. Conclusion Torpedo maculopathy has been described previously as a hypopigmented, nonprogressive lesion of unknown etiology. The findings of global hyperpigmentation, pseudo-lacuna formation, and morphologic changes over time in this lesion challenge these classically held descriptions, and necessitate long-term follow-up with multimodal imaging. PMID:27462244

  2. Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

    PubMed

    Grisham, C M; Mildvan, A S

    1975-01-01

    EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme

  3. Application of high resolution NMR, ESR, and gamma-ray scintillation spectroscopy to the study of ligand binding in proteins. [Torpedo californica

    SciTech Connect

    Lancione, G.V.

    1982-01-01

    Electron spin resonance spectroscopy has been employed to study the nature of the ligand binding site of alpha-1-antitrypsin. Spectra of spin-labeled alpha-1-antitrypsin were recorded at pH's ranging from 2.4 to 12.5. This data demonstrates the tight binding of the spin-label to the protease, and the sensitivity of the bound spin-label to informational changes in the protease inhibitor. A molecular dipstick approach has also been applied to this system and has yielded information on the geometry of the cleft accommodating the spin-label. /sup 160/Terbium(III) exchange experiments have been performed on the acetylcholine receptor protein isolated from Torpedo californica, employing a specially designed flow dialysis apparatus constructed in the laboratory. The apparatus is designed to allow continuous monitoring of /sup 160/Tb(III) gamma-ray emission from the protein compartment of the flow dialysis cell. Nicotinic ligand-induced displacement of /sup 160/Tb(III) from the nicotinic binding site of the receptor was monitored as a funtion of (1) the concentration of nicotinic ligand in the washout buffer, and (2) the nature of the nicotinic ligand in the buffer. Measured /sup 160/Tb(III) exchange half-lives indicate (1) a direct relationship between /sup 160/Tb(III) displacement and nicotinic ligand concentration in the wash-out buffer, and (2) an enhanced /sup 160/Tb(III) displacement for nicotinic agents possessing quaternary ammonium functions.

  4. 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

  5. Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078.

    PubMed

    Tang, Yong; Zhao, Chun-Yan; Tan, Shu-Tang; Xue, Hong-Wei

    2016-08-01

    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

  6. Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M.

    PubMed

    Vasil'ev, V I; Tikhonova, T V; Gvozdev, R I; Tukhvatullin, I A; Popov, V O

    2006-12-01

    The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out. PMID:17223785

  7. α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

    PubMed Central

    Young, Howard S.; Herbette, Leo G.; Skita, Victor

    2003-01-01

    The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. α-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-Å resolution for Torpedo californica nAChR membranes in the absence and presence of α-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-Å lattice spacing between stacked nAChR membrane pairs. In the presence of α-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 Å, and this distance increased to 85 Å in the presence of toxin. Electron density profile structures were calculated in the absence and presence of α-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, α-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer. PMID:12885641

  8. Acetylcholine receptor-specific suppressive T-cell factor from a retrovirally transformed T-cell line.

    PubMed Central

    Sinigaglia, F; Gotti, C; Castagnoli, R; Clementi, F

    1984-01-01

    In both experimental and human myasthenia gravis an impairment in the immune regulation leads to an increased synthesis of antibodies against the nicotinic acetylcholine receptor (AcChoR). The present work reports the establishment of an AcChoR-specific suppressive T-cell line obtained by viral transformation of AcChoR-enriched murine T lymphocytes. Enriched T cells from Torpedo AcChoR-primed mice, prestimulated in vitro with antigen, were infected with radiation leukemia viruses and injected intravenously in congeneic recipient mice. Six months later lymphomas were observed in 20% of the injected mice and two of them, of donor origin, were established as permanent continuous cell lines in vitro. One of these lines, named LA41, expresses Thy-1.2, Lyt-2, and I-Jb surface markers. Culture supernatants of LA41 cells suppress the antigen-specific in vitro proliferation of Torpedo AcChoR-primed lymphocytes. This suppression is antigen-specific since the response induced by fetal calf AcChoR and by other antigens is not affected by addition of LA41 culture supernatant in the proliferative assay. LA41 culture supernatant injected in vivo at the time of antigen-priming suppresses also significantly the production of anti-AcChoR antibodies but not the synthesis of antibodies against other antigens--i.e., fetal calf AcChoR or alpha-bungarotoxin. These data show that LA41 cells constitutively produce Torpedo AcChoR-specific suppressor factor. PMID:6095305

  9. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

    PubMed

    Tabe, Shirou; Hikiji, Hisako; Ariyoshi, Wataru; Hashidate-Yoshida, Tomomi; Shindou, Hideo; Okinaga, Toshinori; Shimizu, Takao; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2016-07-01

    Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons. PMID:27048541

  10. 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.

  11. 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.

  12. 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.

  13. Functional interaction between Lypd6 and nicotinic acetylcholine receptors.

    PubMed

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj; Wang, Hong; Klein, Anders B; Thiriet, Nathalie; Pinborg, Lars H; Muldoon, Pretal P; Wienecke, Jacob; Imad Damaj, M; Kohlmeier, Kristi A; Gondré-Lewis, Marjorie C; Mikkelsen, Jens D; Thomsen, Morten S

    2016-09-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross-linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane-bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine-induced ERK phosphorylation and attenuates nicotine-induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain. PMID:27344019

  14. Purification of the muscarinic acetylcholine receptor from porcine atria.

    PubMed Central

    Peterson, G L; Herron, G S; Yamaki, M; Fullerton, D S; Schimerlik, M I

    1984-01-01

    The muscarinic acetylcholine receptor from porcine atria has been purified 100,000-fold to homogeneity by solubilization in digitonin/cholate and sequential chromatography on wheat germ agglutinin-agarose, diethylaminoethylagarose, hydroxylapatite, and 3-(2'-aminobenzhydryloxy)tropane-agarose. The yield of purified receptor was 4.3% of that found in the membrane fraction, and the purified receptor bound 11.1-12.8 nmol of L-[3H]quinuclidinyl benzilate per mg of protein, corresponding to a binding component Mr of 78,400-90,000. The purified receptor preparation consisted of two polypeptides in approximately equimolar amounts when examined on silver-stained sodium dodecyl sulfate/polyacrylamide gels. The larger polypeptide (Mr 78,000 on 8% polyacrylamide gels) was specifically alkylated with [3H]propylbenzilylcholine mustard, whereas the smaller polypeptide (Mr 14,800) was not labeled. The possibility that the small polypeptide is a contaminant fortuitously appearing in equimolar amounts with the large polypeptide cannot be ruled out at this time. The purified preparation was highly stable, with no measurable change in the number of ligand binding sites or the gel pattern after 1 month's storage on ice. Scatchard analysis showed a single class of binding sites for the antagonist L-[3H]quinuclidinyl benzilate with a dissociation constant of 61 +/- 4 pM. Equilibrium titration experiments demonstrated that the antagonist L-hyoscyamine displaced L-[3H]quinuclidinyl benzilate from a single class of sites (Kd = 475 +/- 30 pM), whereas the agonist carbamoylcholine interacted at two populations of sites (53% +/- 3% high affinity, Kd = 1.1 +/- 0.3 microM; 47% +/- 3% low affinity, Kd = 67 +/- 14 microM). The ligand binding data were very similar to that for the membrane-bound receptor, suggesting that the receptor has not been altered radically during purification. Images PMID:6589642

  15. 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.

  16. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors.

    PubMed

    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(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(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(2)β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC(50)=0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1(2)β1γδ) than for the mouse (α1(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. PMID:23046821

  17. Loss of covalently linked lipid as the mechanism for radiation-induced release of membrane-bound polysaccharide and exonuclease from Micrococcus radiodurans. [/sup 60/CO

    SciTech Connect

    Mitchel, R.E.J.

    1981-08-01

    The mechanism of ..gamma..-radiation-induced release of polysaccharide and exonuclease from the midwall membrane of Micrococcus radiodurans has been examined. These two components appear to be released independently, but by very similar processes. Direct analysis of radiation-released polysaccharide indicated the absence of an alkali-labile neutral lipid normally present in the native material. Radiation-induced release therefore probably results from the radiolytic cleavage of a covalently linked lipid which normally serves to anchor these substances to the membrane. The absence of a natural membrane-bound carotenoid had no effect on the rate of release of these components. Likewise, the absence of exonuclease in an exonuclease minus mutant did not influence the release of polysaccharide. It is suggested that the major pathway of radical transfer from the initiating .OH and culminating in the cleavage of the neutral lipid anchor may not be via the membrane.

  18. 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

  19. 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

  20. Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

    PubMed Central

    Ridley, Helen; Watts, Carys A.; Richardson, David J.; Butler, Clive S.

    2006-01-01

    Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed Vmax of 500 nmol SeO42− min−1 mg−1 (kcat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase. PMID:16885262

  1. 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

  2. Overexpression of membrane-bound fas ligand (CD95L) exacerbates autoimmune disease and renal pathology in pristane-induced lupus.

    PubMed

    Bossaller, Lukas; Rathinam, Vijay A K; Bonegio, Ramon; Chiang, Ping-I; Busto, Patricia; Wespiser, Adam R; Caffrey, Daniel R; Li, Quan-Zhen; Mohan, Chandra; Fitzgerald, Katherine A; Latz, Eicke; Marshak-Rothstein, Ann

    2013-09-01

    Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity. PMID:23918976

  3. Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

    PubMed Central

    Park, Sang Ho; Marassi, Francesca M.; Black, David; Opella, Stanley J.

    2010-01-01

    The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16–A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2–G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state 1H/15N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly. PMID:20816058

  4. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

    PubMed

    Schneider, P; Holler, N; Bodmer, J L; Hahne, M; Frei, K; Fontana, A; Tschopp, J

    1998-04-20

    Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high. PMID:9547332

  5. 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.

  6. Pharmacology of some acetylcholine homologues

    PubMed Central

    Barrass, B. C.; Brimblecombe, R. W.; Rich, P.; Taylor, Joan V.

    1970-01-01

    1. The acetates of several long chain (3 to 12 methylene groups) analogues of choline have been prepared and their pharmacological properties studied. 2. None of the compounds had a high level of activity at the post-ganglionic parasympathetic acetylcholine receptors. The lower members of the series showed weak agonist activity and the homologues with 8 to 10 methylene groups had very weak anticholinergic activity. 3. All the compounds had a depolarizing action at the acetylcholine receptors of the neuromuscular junction and of sympathetic ganglia. At the neuromuscular junction there were two peaks of stimulant activity, one with the hexamethylene and one with the dodecamethylene homologue, whereas at the ganglion there was only one peak, with the hexamethylene homologue. 4. The ganglion-stimulant activity of the higher members of the series was blocked by pretreatment with the anticholinesterase drug dyflos, whereas the activity of lower members was either unaffected by such treatment or slightly potentiated. 5. The results are discussed in terms of possible spatial arrangements of acetylcholine receptor units in the neuromuscular junction and the ganglion. PMID:5420144

  7. Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor

    SciTech Connect

    Revah, F.; Mulle, C.; Pinset, C.; Audhya, T.; Goldstein, G.; Changeux, J.P.

    1987-05-01

    The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C/sub 2/ myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (> 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca/sup 2 +/-insensitive manner. At lower concentrations (2 nM), Tpo applied on C/sub 2/ myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca/sup 2 +/ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca/sup 2 +/ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca/sup 2 +/ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker (/sup 3/H)phencyclidine, enhances, at low concentrations, the binding of (/sup 3/H)acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid ..beta..-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca/sup 2 +/, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state.

  8. 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.

  9. 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…

  10. 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