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1

Theoretical Investigation of the Enzymatic Phosphoryl Transfer of ?-phosphoglucomutase: Revisiting Both Steps of the Catalytic Cycle  

SciTech Connect

Enzyme catalyzed phosphate transfer is a part of almost all metabolic processes. Such reactions are of central importance for the energy balance in all organisms and play important roles in cellular control at all levels. Mutases transfer a phosphoryl group while nucleases cleave the phosphodiester linkages between two nucleotides. The subject of our present study is the Lactococcus lactis ?-phosphoglucomutase (?-PGM), which effectively catalyzes the interconversion of ?-D-glucose-1-phosphate (?-G1P) to ?- D-glucose-6-phosphate (?-G6P) and vice versa via stabile intermediate ?-D-glucose-1,6-(bis)phosphate (?-G1,6diP) in the presence of Mg2+. In this paper we revisited the reaction mechanism of the phosphoryl transfer starting from the bisphosphate ?-G1,6diP in both directions (toward ?-G1P and ?-G6P) combining docking techniques and QM/MM theoretical method at the DFT/PBE0 level of theory. In addition we performed NEB (nudged elastic band) and free energy calculations to optimize the path and to identify the transition states and the energies involved in the catalytic cycle. Our calculations reveal that both steps proceed via dissociative pentacoordinated phosphorane, which is not a stabile intermediate but rather a transition state. In addition to the Mg2+ ion, Ser114 and Lys145 also play important roles in stabilizing the large negative charge on the phosphate through strong coordination with the phosphate oxygens and guiding the phosphate group throughout the catalytic process. The calculated energy barrier of the reaction for the ?-G1P to ?-G1,6diP step is only slightly higher than for the ?-G1,6diP to ?-G6P step (16.10 kcal mol-1 versus 15.10 kcal mol-1) and is in excellent agreement with experimental findings (14.65 kcal mol-1).

Elsasser, Brigitta M.; Dohmeier-Fischer, Silvia; Fels, Gregor

2012-07-12

2

Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia coli Alkaline Phosphatase † ‡  

Microsoft Academic Search

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia

Patrick J. O’Brien; Jonathan Kyle Lassila; Timothy D. Fenn; Jesse G. Zalatan; Daniel Herschlag

2008-01-01

3

Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase  

SciTech Connect

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by {approx}3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 {angstrom} X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

2009-05-22

4

Biochemical Control of CARM1 Enzymatic Activity by Phosphorylation*  

PubMed Central

Coactivator-associated arginine methyltransferase 1 (CARM1) is a dual functional coregulator that facilitates transcription initiation by methylation of Arg17 and Arg26 of histone H3 and also dictates the subsequent coactivator complex disassembly by methylation of the steroid receptor coactivator family coactivators and p300/cAMP-response element-binding protein-binding protein. However, the regulation of CARM1 enzymatic activity and substrate specificity remains largely unknown. In this study, we report that CARM1 function is regulated by phosphorylation at Ser217, a residue completely conserved in the type I protein arginine methyltransferase (PRMT) family of enzymes. Comparative analysis of the published CARM1 crystal structures reveals that the hydroxyl group of Ser217 forms a strong hydrogen bond with the carbonyl oxygen atom of Tyr154 to lock the cofactor S-adenosylmethionine inside the binding cavity. Phosphorylation of Ser217 disrupts this hydrogen bond and subsequently abolishes S-adenosylmethionine binding and its methyltransferase activity. Importantly, Tyr154 is also conserved in the type I PRMT family of enzymes, suggesting a general role of this hydrogen bond in maintaining the holo structure of the type I PRMT catalytic domain. Moreover, we found that phosphorylation at Ser217 also promoted CARM1 cytoplasmic localization and that this translocation occurred mainly during mitosis. We propose that phosphorylation at Ser217 serves as a molecular switch for controlling CARM1 enzymatic activity during the cell cycle.

Feng, Qin; He, Bin; Jung, Sung-Yun; Song, Yongcheng; Qin, Jun; Tsai, Sophia Y.; Tsai, Ming-Jer; O'Malley, Bert W.

2009-01-01

5

Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis  

PubMed Central

Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

Lassila, Jonathan K.; Zalatan, Jesse G.; Herschlag, Daniel

2012-01-01

6

Metallic fluoride complexes as phosphate analogues for structural and mechanistic studies of phosphoryl group transfer enzymes.  

PubMed

There have been intensive efforts to try to understand the details of phosphoryl transfer reactions extending from nonenzymatic (or enzyme model) systems to the mechanisms of the enzyme catalysed reactions. As phosphate analogues, few metallic fluorides AlFx, BeFx and MgFx affect the activity of a variety of phosphoryl transfer enzymes, and it is accepted that these small inorganic complexes are useful chemical probes for structural and mechanistic studies in enzymology because they are able to mimic phosphoryl group in ground state (BeFx) as well as in transition state (AlFx,MgFx). Al3+ and Be2+ tend to form stable complexes with different fluoride anions (x = 1 to 4) spontaneously in aqueous solution but Mg2+ does not. BeFx geometry is strictly tetrahedral resembling the phosphate ground state when bound to an acyl group of protein active site (phosphorylated acyl groups are unstable otherwise), or the Michaelis complex when BeFx concominantly with nucleoside diphosphate replaces g-phosphate group in nucleoside triphosphate sites. AlFx and MgFx are identified as enzymatic analogues of phosphoryl transition state where both are able to form different coordination geometries within the enzyme active sites: trigonal bipyramidal (AlF3 and MgF3-) or octahedral (AlF4- or MgF42-). The geometry and charge of MgF3- are the best suited to mimicking the trigonal planar PO3- moiety of phosphoryl transfer transition state but MgF3- does not, unlike aluminum and beryllium fluoride complexes, exists in solution and can be assembled and stabilized in suitable active site only. Therefore it is particularly interesting to characterize as a potentially highly accurate transition state analogue and may be the best reagent of choice for studying phosphoryl transfer reactions in future. PMID:24061722

Goli?nik, Marko

2010-06-01

7

Mapping Sites of Protein Phosphorylation by Mass Spectrometry Utilizing a Chemical-Enzymatic Approach: Characterization of Products from ?-S1Casein Phosphopeptides  

PubMed Central

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI–TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine ?-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented.

McCormick, Daniel J.; Holmes, Michael W.; Muddiman, David C.; Madden, Benjamin J.

2008-01-01

8

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase.  

PubMed

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10(10)-fold: but how? Our structures of the yeast PPase product complex at 1.15 A and fluoride-inhibited complex at 1.9 A visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O(nu)) directly attacks PPi with a pK(a) vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O(nu), in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography. PMID:11248042

Heikinheimo, P; Tuominen, V; Ahonen, A K; Teplyakov, A; Cooperman, B S; Baykov, A A; Lahti, R; Goldman, A

2001-03-13

9

Phospholipid transfer protein (PLTP) reduces phosphorylation of tau in human neuronal cells (HCN2)  

PubMed Central

Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer’s disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau, and increases levels of the inactive form of glycogen synthase kinase-3? (GSK3?) in HCN2 cells. Furthermore, inhibition of the phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3? phosphorylated at serine 9 (pGSK3?Ser9) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), since PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses PLTP-induced increase in levels of phosphorylated Akt (pAktThr308 and pAktSer473), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons.

Dong, Weijiang; Albers, John J.; Vuletic, Simona

2009-01-01

10

Studies on the mechanisms of phosphorothioyl transfer in enzymatic and nonenzymatic reactions  

Microsoft Academic Search

The stereochemical course of rabbit liver fructose bisphosphatase (E.C.3.1.3.11) was determined by hydrolyzing a substrate analog (Sp)-(1-¹⁸O)-fructose 1-phosphorothioate 6-phosphate in Hâ¹⁷O, incorporating the chiral, inorganic phosphorothioate product into ATP..beta..S, and analyzing the isotopic distribution of ¹⁸O in ATP..beta..S by ³¹P NMR. The results indicate that the 1-phosphoryl group is transferred with inversion of configuration. A series of ¹⁸O-enrichment and exchange,

Domanico

1986-01-01

11

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue  

NASA Astrophysics Data System (ADS)

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Lanucara, Francesco; Chi Hoo Lee, Dave; Eyers, Claire E.

2013-12-01

12

Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.  

PubMed

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. PMID:24297471

Lanucara, Francesco; Lee, Dave Chi Hoo; Eyers, Claire E

2014-02-01

13

A redox beginning: Which came first phosphoryl, acyl, or electron transfer ?. [Abstract only  

NASA Technical Reports Server (NTRS)

Thermodynamic and kinetic information available on the synthesis of prebiotic monomers and polymers will be examined in order to illuminate the prebiotic plausibility of polymer syntheses based on (a) phosphoryl transfer that yields phosphodiester polymers, (b) acyl transfer that gives polyamides, and (c) electron transfer that produces polydisulfide or poly(thio)ester polymers. New experimental results on the oxidative polymerization of 2,3-dimercaptopropanol by ferric ions on the surface of ferric hydroxide oxide will be discussed as a chemical model of polymerization by electron transfer. This redox polymerization that yields polymers with a polydisulfide backbone was found to give oligomers up to the 15-mer from 1 mM of 2,3-dimercaptopropanol after one day at 25 C. High pressure liquid chromatography (HPLC) analysis of the oligomers was carried out on an Alltech OH-100 column eluted with acetonitrile-water.

Weber, Arthur L.

1994-01-01

14

The Phosphoryl Transfer Domain of UhpB Interacts with the Response Regulator UhpA  

PubMed Central

Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription. UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT. Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione S-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of uhpT expression in a uhp+ strain. This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation. The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation. Point mutations which allow GST-Bc to activate uhpT expression or which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain. The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR. The GST-Bc protein accelerated dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313. Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred. Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA.

Wright, Jesse S.; Kadner, Robert J.

2001-01-01

15

Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides  

PubMed Central

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.

Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffmann, Ralf; van de Goor, Tom; Metz, Thomas O.; Smith, Richard D.

2009-01-01

16

Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides  

SciTech Connect

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in diabetes mellitus research, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.

Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffman, Ralf; van der Goor, Tom; Metz, Thomas O.; Smith, Richard D.

2007-03-15

17

Studies on the mechanisms of phosphorothioyl transfer in enzymatic and nonenzymatic reactions  

SciTech Connect

The stereochemical course of rabbit liver fructose bisphosphatase (E.C.3.1.3.11) was determined by hydrolyzing a substrate analog (Sp)-(1-/sup 18/O)-fructose 1-phosphorothioate 6-phosphate in H/sub 2//sup 17/O, incorporating the chiral, inorganic phosphorothioate product into ATP..beta..S, and analyzing the isotopic distribution of /sup 18/O in ATP..beta..S by /sup 31/P NMR. The results indicate that the 1-phosphoryl group is transferred with inversion of configuration. A series of /sup 18/O-enrichment and exchange, single-turnover experiments rule out an enzyme acylphosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water. To determine whether a monomeric metaphosphorothioate intermediate is formed during the hydrolysis of phosphorothioate monoesters, the stereochemical course of the hydrolysis of chiral (Rp)- and (Sp)-(/sup 18/O) O-p-nitrophenyl phosphorothioate in H/sub 2//sup 17/O was determined as per rabbit liver fructose bisphosphatase. /sup 31/P NMR analysis of the isotopic distribution of the ..beta..-P species of (Rp)-(..beta..-/sup 17/O,..beta..-/sup 18/O) ATP..beta..S for both isomers showed the monoester hydrolysis to proceed with inversion of configuration implying that if the metaphosphorothioate intermediate was formed, it did not exist long enough to become symmetrically solvated and, subsequently, suffered nucleophilic attack on the face opposing that occupied by the leaving group, p-nitrophenol.

Domanico, P.L.

1986-01-01

18

In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate.  

PubMed

A polyhydroxyalkanoate (PHA) was enzymatically synthesized in vitro, and the end structure of PHA associated with a chain transfer (CT) reaction was investigated. In the CT reaction, PHA chain transfers from PHA synthase (PhaC) to a CT agent, resulting in covalent bonding of CT agent to the PHA chain at its carboxyl end. In vitro CT reaction has never been demonstrated because of relatively low yields of in vitro synthesized poly[(R)-3-hydroxybutyrate)] (P(3HB)), which makes it difficult to characterize the end structures of the polymers by nuclear magnetic resonance (NMR). To overcome these difficulties, a novel in vitro synthesis method that produced relatively larger amounts of P(3HB) was developed by employing PhaCDa from Delftia acidovorans and two enantioselective enoyl-coenzyme A (CoA) hydratases which were R-hydratase (PhaJAc) from Aeromonas caviae and S-hydratase (FadB1x) from Pseudomonas putida KT2440 with ?-butyrolactone and CoA as starting materials. Using this method, P(3HB) synthesis was performed with tetraethylene glycols (TEGs) as a discriminable CT agent, and the resultant P(3HB) was characterized by (1)H-NMR. NMR analysis revealed that the carboxylic end of P(3HB) was covalently linked to TEGs, providing the first direct evidence of in vitro CT reaction. PMID:23474615

Tomizawa, Satoshi; Sato, Shun; Lan, John Chi-Wei; Nakamura, Yoshiyuki; Abe, Hideki; Tsuge, Takeharu

2013-06-01

19

Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.  

PubMed

A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250?Acm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60?Wcm(-2), while with ethanol 40?Wcm(-2) was obtained. PMID:24953844

Arrocha, Andrés A; Cano-Castillo, Ulises; Aguila, Sergio A; Vazquez-Duhalt, Rafael

2014-11-15

20

Molecular Basis of 1,6-Anhydro Bond Cleavage and Phosphoryl Transfer by Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase*  

PubMed Central

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the ? phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.

Bacik, John-Paul; Whitworth, Garrett E.; Stubbs, Keith A.; Yadav, Anuj K.; Martin, Dylan R.; Bailey-Elkin, Ben A.; Vocadlo, David J.; Mark, Brian L.

2011-01-01

21

Asymmetric phosphorylation through catalytic P(III) phosphoramidite transfer: Enantioselective synthesis of d-myo-inositol-6-phosphate  

PubMed Central

Despite the ubiquitous use of phosphoramidite chemistry in the synthesis of biophosphates, catalytic asymmetric phosphoramidite transfer remains largely unexplored for phosphate ester synthesis. We have discovered that a tetrazole-functionalized peptide, in the presence of 10-? molecular sieves, functions as an enantioselective catalyst for phosphite transfer. This chemistry in turn has been used as the key step in a streamlined synthesis of myo-inositol-6-phosphate. Mechanistic insights implicate phosphate as a directing group for a highly selective kinetic resolution of a protected inositol monophosphate. This work represents a distinct and efficient method for the selective catalytic phosphorylation of natural products.

Jordan, Peter A.; Kayser-Bricker, Katherine J.; Miller, Scott J.

2010-01-01

22

Protein Kinase D Negatively Regulates Hepatitis C Virus Secretion through Phosphorylation of Oxysterol-binding Protein and Ceramide Transfer Protein*  

PubMed Central

Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser132 and Ser240 residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process.

Amako, Yutaka; Syed, Gulam H.; Siddiqui, Aleem

2011-01-01

23

Physical Properties and Enzymatic Digestibility of Phosphorylated ae, wx , and Normal Maize Starch Prepared at Different pH Levels 1  

Microsoft Academic Search

Cereal Chem. 76(6):938-943 Phosphorylated starches were prepared with sodium tripolyphosphate (STPP) at pH 6, 8, and 10 from waxy (wx, 3.3% amylose), normal (22.4% amylose), and two high-amylose (ae, 47 and 66% amylose) maize starches. After phosphorylation, the gelatinization peak temperature ( Tp) decreased and pasting peak viscosity (PV) increased for all the starches except wx, which showed a slight

Huijun Liu; Lawrence Ramsden; Harold Corke

1999-01-01

24

Two and Three-dimensional Gene Transfer from Enzymatically Degradable Hydrogel Scaffolds  

PubMed Central

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation. In this report, we explored gene transfer to MSCs seeded on top, or inside matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP degradable peptides via Michael Addition chemistry. Gene transfer was visualized and quantified through using a vector encoding for green fluorescent protein and luciferase. We found that gene transfer to MSCs was possible for cells seeded both in two and three dimensions. The amount of luciferase expression was similar for cells seeded in two and three dimensions even though the number of cells in three dimensions is significantly higher, indicating that gene transfer to cells seeded in two dimensions is more efficient than for cells seeded in three dimensions. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.

Lei, Yuguo; Ng, Quinn K. T.; Segura, Tatiana

2010-01-01

25

Two and three-dimensional gene transfer from enzymatically degradable hydrogel scaffolds.  

PubMed

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation. In this report, we explored gene transfer to MSCs seeded on top or inside matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP degradable peptides via Michael Addition chemistry. Gene transfer was visualized and quantified through using a vector encoding for green fluorescent protein and luciferase. We found that gene transfer to MSCs was possible for cells seeded both in two and three dimensions. The amount of luciferase expression was similar for cells seeded in two and three dimensions even though the number of cells in three dimensions is significantly higher, indicating that gene transfer to cells seeded in two dimensions is more efficient than for cells seeded in three dimensions. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long-lasting signals in vivo, which are essential for the regeneration of functional tissues. PMID:20232458

Lei, Yuguo; Ng, Quinn K T; Segura, Tatiana

2010-09-01

26

Small electron-transfer proteins as mediators in enzymatic electrochemical biosensors.  

PubMed

Electrochemical mediators transfer redox equivalents between the active sites of enzymes and electrodes and, in this way, initiate bioelectrocatalytic redox processes. This has been very useful in the development of the so-called second-generation biosensors, in which they transduce a catalyzed reaction into an electrical signal. Among other pre-requisites, redox mediators must be readily oxidized and/or reduced at the electrode surface and readily interact with the biorecognition component. Small chemical compounds (e.g. ferrocene derivatives, ruthenium, or osmium complexes and viologens) are frequently used for this purpose but, lately, small redox proteins (e.g. horse heart cytochrome c) have also been used as redox partners in biosensing applications. In general, docking between two complementary proteins introduces a second level of selectivity to the biosensor and enlarges the list of compounds analyzed. Moreover, electrochemical interferences are frequently minimized owing to the small overpotentials achieved. This paper provides an overview of enzyme biosensors that are mediated by electron-transfer proteins. The paper begins with a brief discussion of mediated electrochemistry in biosensing systems and proceeds with a detailed description of relevant work on the cooperative use of redox enzymes and biological electron donors and/or acceptors. PMID:23430181

Silveira, Célia M; Almeida, M Gabriela

2013-04-01

27

Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect  

PubMed Central

Phosphomannomutase/phosphoglucomutase contributes to infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. NMR spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. 13C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 ?-helices, C-capping motifs, and 20 of the 22 ?-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These are attributable to the phosphorylation state and possibly to conformational interconversions. S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose-1-phosphate substrate by impairment of kcat. Addition of the glucose-1,6-bisphosphate intermediate accelerated this reaction by 2 – 3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft, while preserving the secondary structure in solution. Diminished peak heights and faster 15N relaxation are suggestive of line broadening and millisecond fluctuations within four loops that can contact phosphosugars. 15N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1 to 3, and with a different principal axis of diffusion. This adds to the crystallographic evidence for domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of intermediate, substrate binding, and product release.

Sarma, Akella V.S.; Anbanandam, Asokan; Kelm, Allek; Mehra-Chaudhary, Ritcha; Wei, Yirui; Qin, Peiwu; Lee, Yingying; Berjanskii, Mark V.; Mick, Jacob A.; Beamer, Lesa J.; Van Doren, Steven R.

2012-01-01

28

Enrichment and Analysis of Non-enzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron Transfer Dissociation Mass Spectrometry  

PubMed Central

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.

Zhang, Qibin; Tang, Ning; Brock, Jonathan W. C.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John W.; Smith, Richard D.; Metz, Thomas O.

2008-01-01

29

Cardiac mitochondrial matrix and respiratory complex protein phosphorylation  

PubMed Central

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain ?-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria.

Covian, Raul

2012-01-01

30

An enzymatic bridge between carbohydrate and amino acid metabolism: regulation of glutamate dehydrogenase by reversible phosphorylation in a severe hypoxia-tolerant crayfish.  

PubMed

Glutamate dehydrogenase (GDH) (EC 1.4.1.3) is a crucial enzyme involved in bridging two metabolic pathways, gating the use of glutamate for either amino acid metabolism, or carbohydrate metabolism. The present study investigated GDH from tail muscle of the freshwater crayfish Orconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20-h severe hypoxic). Evidence indicated that GDH was converted to a high phosphate form under oxygen limitation. ProQ Diamond phosphoprotein staining showed a 42% higher bound phosphate content on GDH from muscle of severely hypoxic crayfish compared with the aerobic form, and treatment of this GDH with commercial phosphatase (alkaline phosphatase), and treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1 + PP2A, PP2B, and PP2C) yielded significant increases in the fold activation by ADP of GDH from both control and severe hypoxic conditions. By contrast, stimulation of the activities of endogenous protein kinases (AMPK, PKA or CaMK) significantly reduced the ADP fold activation from control animals. The physiological consequence of severe hypoxia-induced GDH phosphorylation may be to suppress GDH activity under low oxygen, shutting off this critical bridge point between two metabolic pathways. PMID:22076534

Dawson, Neal J; Storey, Kenneth B

2012-04-01

31

Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative.  

PubMed

The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities. PMID:1718435

Donella-Deana, A; Varro, A; Dockray, G J; Pinna, L A

1991-10-16

32

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.  

PubMed

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride. PMID:18318536

Baxter, Nicola J; Blackburn, G Michael; Marston, James P; Hounslow, Andrea M; Cliff, Matthew J; Bermel, Wolfgang; Williams, Nicholas H; Hollfelder, Florian; Wemmer, David E; Waltho, Jonathan P

2008-03-26

33

Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay.  

PubMed

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KDeltaCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (Km) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4+/-0.29 and 0.9+/-0.48 microM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors. PMID:16213157

Zhang, Wei-Guo; Shor, Boris; Yu, Ker

2006-04-01

34

Enzymatic regeneration of adenosine triphosphate cofactor  

NASA Technical Reports Server (NTRS)

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Marshall, D. L.

1974-01-01

35

Insights into the Phosphoryl Transfer Mechanism of Cyclin-dependent Protein Kinases from Ab Initio QM/MM Free-energy Studies  

PubMed Central

Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensible role in cellular signaling, and their malfunctioning is implicated in many diseases. A better understanding of the catalytic mechanism will help design novel and effective mechanism-based inhibitors of these enzymes. In this work, ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a dissociative, metaphosphate-like structure, stabilized by the Mg2+ ion and several hydrogen bonds. The calculated free-energy barrier is consistent with experimental values. Implications of our results in this and other protein kinases are discussed.

Smith, Gregory K.; Ke, Zhihong; Hengge, Alvan C.

2011-01-01

36

Computer simulations of enzymatic reactions: examination of linear free-energy relationships and quantum-mechanical corrections in the initial proton-transfer step of carbonic anhydrase.  

PubMed

Computer simulation approaches can provide a powerful tool for correlating the structure of enzymes with their catalytic activity. One of the most effective ways of simulating enzymatic reactions is provided by the empirical valence bond method. The general applicability of this method has been demonstrated in several enzymatic reactions and it is reexamined here in a study of the initial proton-transfer step in the catalytic reaction of carbonic anhydrase. The simulations produce a rate constant which is in agreement with the observed kinetic data and emphasizes the importance of the electrostatic effect associated with the catalytic zinc ion. The calculations are also used to examine the validity of linear free-energy relationships (LFERs) in enzyme catalysis and to evaluate quantum-mechanical corrections of the calculated rate constant. It is found that LFERs are valid in the present case and it is argued that this reflects the fact that the protein responds linearly to the development of electrostatic forces during the reaction. It is concluded that the present approach can be used to augment experimental studies in establishing the general validity of LFERs. It is noted, however, that such relationships are much more valid for transitions between different resonance structures than for transitions between reactants and product states. PMID:1337846

Warshel, A; Hwang, J K; Aqvist, J

1992-01-01

37

Differential expression of a C-terminal splice variant of phosphatidylinositol transfer protein ? lacking the constitutive-phosphorylated Ser262 that localizes to the Golgi compartment  

PubMed Central

Mammalian PITP? (phosphatidylinositol transfer protein ?) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITP? is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITP? (PITP?-sp2) where the C-terminal 15 amino acids of PITP?-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITP?-sp1 is the product of the first 11 exons, whereas PITP?-sp2 is a product of the first 10 exons followed by the twelfth exon – exon 11 being ‘skipped’. Both splice variants are capable of PtdIns and PtdCho transfer, with PITP?-sp2 being unable to transport SM. PITP? is ubiquitously expressed, with the highest amounts of PITP? found in HL60 cells and in rat liver; HL60 cells express only PITP?-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITP?-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITP?-sp1 are present. In contrast, PITP?-sp2 lacks the constitutively phosphorylated Ser262 (replaced with glutamine). Nonetheless, both PITP? variants localize to the Golgi and, moreover, dephosphorylation of Ser262 of PITP?-sp1 does not affect its Golgi localization. The presence of PITP? sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITP? gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

Morgan, Clive P.; Allen-Baume, Victoria; Radulovic, Marko; Li, Michelle; Skippen, Alison; Cockcroft, Shamshad

2006-01-01

38

Identification of a potential general acid/base in the reversible phosphoryl transfer reactions catalyzed by tyrosine recombinases: Flp H305  

PubMed Central

Summary Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate, similar to the mechanism of type Ib topoisomerases [1]. Unlike most related enzymes, Flp’s nucleophilic tyrosine derives from a different protomer than the remainder of its active site [2, 3]. Because the tyrosine can be supplied exogenously, non-natural synthetic analogs can be used. Here we examine the catalytic role of Flp’s conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa?10), or 3-fluoro-tyrosine (pKa?8.4). Religation was studied using DNA substrates with 3’-phospho-cresol (pKa?10) or 3’-para-nitro-phenol (pKa?7.1) mimicking the covalent protein-DNA intermediate. In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide the first experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases.

Whiteson, Katrine L.; Chen, Yu; Chopra, Neeraj; Raymond, Amy C.; Rice, Phoebe A.

2007-01-01

39

Biofuel cells: enhanced enzymatic bioelectrocatalysis.  

PubMed

Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods. PMID:22524222

Meredith, Matthew T; Minteer, Shelley D

2012-01-01

40

Aromatase is phosphorylated in situ at Serine-118  

PubMed Central

Phosphorylation of the cytochrome P450 aromatase has been proposed as a switch to rapidly modulate enzymatic activity and estrogen biosynthesis. Herein, we demonstrate that aromatase serine-118 is a potential phosphorylation site in mammalian cells. The amino acid context surrounding S118 is highly conserved among diverse animal species and suggests that an AGC-like kinase may phosphorylate aromatase. Mutation of S118 to Ala blocked phosphorylation. Mutation of S118 to either Ala or Asp destabilized aromatase, indicating an important structural role for S118. The phosphomimetic S118D mutant showed decreased specific enzymatic activity, decreased Vmax, and increased Km, while the S118A phospho-inhibiting mutant showed opposite effects. Our findings suggest that phosphorylation of S118 may decrease aromatase activity, presenting a mechanism whereby kinase signaling may modulate estrogen production and hormone balance.

Miller, Todd W.; Shin, Incheol; Kagawa, Norio; Evans, Dean B.; Waterman, Michael R.; Arteaga, Carlos L.

2010-01-01

41

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells  

PubMed Central

SUMMARY Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs.

Huang, Ying; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin

2013-01-01

42

Influence of mass transfer limitations on the enzymatic synthesis of beta-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose.  

PubMed

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products. PMID:18040724

Gonçalves, L R B; Ferreira, A L O; Fernandez-Lafuente, R; Guisan, J M; Giordano, R C; Giordano, R L C

2008-08-01

43

Enzymatic synthesis of farnesyl laurate in organic solvent: initial water activity, kinetics mechanism, optimization of continuous operation using packed bed reactor and mass transfer studies.  

PubMed

The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V (max) = 5.80 mmol l(-1) min(-1) g enzyme(-1), K (m,A) = 0.70 mmol l(-1) g enzyme(-1), K (m,B) = 115.48 mmol l(-1) g enzyme(-1), K (i) = 11.25 mmol l(-1) g enzyme(-1). The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07 ± 0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited. PMID:21327986

Rahman, N K; Kamaruddin, A H; Uzir, M H

2011-08-01

44

Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells  

SciTech Connect

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China) [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

2010-07-02

45

Enzymatic mechanism of human apurinic/apyrimidinic endonuclease against a THF AP site model substrate†  

PubMed Central

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 105 potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double stranded DNA oligomers containing tetrahydrofuran (THF) with a 5’ phosphorothioate linkage as the abasic site substrate. Using H218O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one-step associative phosphoryl transfer mechanism on a THF-containing substrate.

Mundle, Sophia T.; Delaney, James C.; Essigmann, John M.; Strauss, Phyllis R.

2009-01-01

46

Effect of ageing and ischemia on enzymatic activities linked to Krebs' cycle, electron transfer chain, glutamate and aminoacids metabolism of free and intrasynaptic mitochondria of cerebral cortex.  

PubMed

The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs' cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (Vmax) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I-III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate-oxaloacetate-and glutamate-pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light mitochondria seems to be more affected in aged animals. These data allow the hypothesis that the observed modifications of catalytic activities in non-synaptic and intra-synaptic mitochondrial enzyme systems linked to energy metabolism, amino acids and glutamate metabolism are primary responsible for the physiopathological responses of cerebral tissue to complete cerebral ischemia for 15 min duration during ageing. PMID:19495970

Villa, Roberto Federico; Gorini, Antonella; Hoyer, Siegfried

2009-12-01

47

The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu  

PubMed Central

Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

De Gieter, Steven; van Nuland, Nico A.J.; Loris, Remy; Zenkin, Nikolay

2013-01-01

48

Enzymatic Template Polymerization.  

National Technical Information Service (NTIS)

A conductive polymer is formed enzymatically in the presence of a polynucleotide template. The method includes combining at least one redox monomer with a polynucleotide template and a redox enzyme, such as horseradish peroxidase, to form a reaction mixtu...

L. A. Samuelson F. Bruno S. K. Tripathy S. Tripathy R. Nagarajan J. Kumar W. Liu

2004-01-01

49

The biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro.  

PubMed

A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain. PMID:9694888

Yuan, Y; Mead, D; Schroeder, B G; Zhu, Y; Barry, C E

1998-08-14

50

Enzymatic desulfurization of coal  

SciTech Connect

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))

1988-12-15

51

Enzymatic desulfurization of coal  

SciTech Connect

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes as well as commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V. (Holometrix, Inc., Cambridge, MA (United States)); Marquis, J.K. (Boston Univ., MA (United States). School of Medicine)

1989-06-16

52

Phosphorylation of Thylakoid Proteins  

Microsoft Academic Search

Application of novel techniques for the characterization of in vivo protein phosphorylation has revealed sixteen distinct\\u000a phosphorylation sites in ten integral and two peripheral proteins in photosynthetic thylakoid membranes. In addition to phosphorylation\\u000a of the photosystem II (PS II) proteins D1, D2, CP43, and PsbH, and the light-harvesting antenna polypeptides LHCII and CP29,\\u000a phosphorylation has been found in photosystem I

Alexander V. Vener

53

Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation  

PubMed Central

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the ?-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.

Schmid, Thomas Ernst; Zlobinskaya, Olga; Multhoff, Gabriele

2012-01-01

54

Substrate-dependent control of MAPK phosphorylation in vivo.  

PubMed

Phosphorylation of the mitogen-activated protein kinase (MAPK) is essential for its enzymatic activity and ability to control multiple substrates inside a cell. According to the current models, control of MAPK phosphorylation is independent of its substrates, which are viewed as mere sensors of MAPK activity. Contrary to this modular view of MAPK signaling, our studies in the Drosophila embryo demonstrate that substrates can regulate the level of MAPK phosphorylation in vivo. We demonstrate that a twofold change in the gene dosage of a single substrate can induce a significant change in the phosphorylation level of MAPK and in the conversion of other substrates. Our results support a model where substrates of MAPK counteract its dephosphorylation by phosphatases. Substrate-dependent control of MAPK phosphorylation is a manifestation of a more general retroactive effect that should be intrinsic to all networks with covalent modification cycles. PMID:21283143

Kim, Yoosik; Paroush, Ze'ev; Nairz, Knud; Hafen, Ernst; Jiménez, Gerardo; Shvartsman, Stanislav Y

2011-02-01

55

Substrate-dependent control of MAPK phosphorylation in vivo  

PubMed Central

Phosphorylation of the mitogen-activated protein kinase (MAPK) is essential for its enzymatic activity and ability to control multiple substrates inside a cell. According to the current models, control of MAPK phosphorylation is independent of its substrates, which are viewed as mere sensors of MAPK activity. Contrary to this modular view of MAPK signaling, our studies in the Drosophila embryo demonstrate that substrates can regulate the level of MAPK phosphorylation in vivo. We demonstrate that a twofold change in the gene dosage of a single substrate can induce a significant change in the phosphorylation level of MAPK and in the conversion of other substrates. Our results support a model where substrates of MAPK counteract its dephosphorylation by phosphatases. Substrate-dependent control of MAPK phosphorylation is a manifestation of a more general retroactive effect that should be intrinsic to all networks with covalent modification cycles.

Kim, Yoosik; Paroush, Ze'ev; Nairz, Knud; Hafen, Ernst; Jimenez, Gerardo; Shvartsman, Stanislav Y

2011-01-01

56

cPLA2 is phosphorylated and activated by MAP kinase  

Microsoft Academic Search

Treatment of cells with agents that stimulate the release of arachidonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). Here we report that cPLA2 is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLA2 phosphorylation by MAP kinase, Ser-505, is identical to the

Lih-Ling Lin; Markus Wartmann; Alice Y. Lin; John L. Knop; Alpna Seth; Roger J. Davis

1993-01-01

57

Phosphorylation states of phospholamban.  

PubMed

Phospholamban is a small integral membrane protein of cardiac, smooth, and slow-twitch skeletal muscle sarcoplasmic reticulum that interacts with the Ca2+ pump of these organelles and inhibits Ca(2+)-pump activity while in the dephosphorylated form. Three sites of Ser/Thr phosphorylation have been identified in the primary sequence of phospholamban, at Ser-10, Ser-16, and Thr-17. In vitro studies indicate that these residues are phosphorylated by PKC (Ser-10), PKA, PKG or PKC (Ser-16), and CaM kinase II (Thr-17). Phosphorylation of Ser-16 (or Thr-17) is accompanied by an increase in Ca2+ pump activity in direct proportion to the stoichiometry of phosphorylation. Dual phosphorylation of both Ser-16 and Thr-17 does not cause any further stimulation of pump function over that achieved by stoichiometric phosphorylation of a single site. Examination of the pattern of phosphorylation in vivo has been aided by the generation of polyclonal antibodies specific for the phosphorylated forms of phospholamban. beta-Adrenergic stimulation of cardiac muscle results in phosphorylation of both Ser-16 and Thr-17. The time course of Ser-16 phosphorylation precedes Thr-17. The spatial distribution of Ser-16 and Thr-17 phosphorylated forms of phospholamban is not identical; phospholamban located in the nuclear membrane of a cardiac myocyte is phosphorylated exclusively on Ser-16, whereas phospholamban molecules in the SR membrane of the same cell are phosphorylated on Ser-16 and/or Thr-17. Finally, we have identified a novel stimulus for the phosphorylation of phospholamban. Ca2+ store depletion, achieved by exposure of myocytes to SERCA inhibitors, prompts the phosphorylation of phospholamban on Ser-16. This would be expected to increase Ca2+ uptake by the SR in an attempt to achieve the refilling of the SR. PMID:10603938

Colyer, J

1998-09-16

58

SOLID-PHASE ASSAY FOR THE PHOSPHORYLATION OF PROTEINS BLOTTED ON NITROCELLULOSE MEMBRANE FILTERS  

EPA Science Inventory

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters and the blotted polypeptides are phosphorylated with ...

59

Enzymatic repair of Amadori products.  

PubMed

Protein deglycation, a new form of protein repair, involves several enzymes. Fructosamine-3-kinase (FN3K), an enzyme found in mammals and birds, phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins. This enzyme acts particularly well on fructose-epsilon-lysine, both in free form and in the accessible regions of proteins. Mice deficient in FN3K accumulate protein-bound fructosamines and free fructoselysine, indicating that the deglycation mechanism initiated by FN3K is operative in vivo. Mammals and birds also have an enzyme designated 'FN3K-related protein' (FN3KRP), which shares ? 65% sequence identity with FN3K. Unlike FN3K, FN3KRP does not phosphorylate fructosamines, but acts on ribulosamines and erythrulosamines. As with FN3K, the third carbon is phosphorylated and this leads to destabilization of the ketoamines. Experiments with intact erythrocytes indicate that FN3KRP is also a protein-repair enzyme. Its physiological substrates are most likely formed from ribose 5-phosphate and erythrose 4-phosphate, which give rise to ketoamine 5- or 4-phosphates. The latter are dephosphorylated by 'low-molecular-weight protein-tyrosine-phosphatase-A' (LMW-PTP-A) before FN3KRP transfers a phosphate on the third carbon. The specificity of FN3K homologues present in plants and bacteria is similar to that of mammalian FN3KRP, suggesting that deglycation of ribulosamines and/or erythrulosamines is an ancient mechanism. Mammalian cells contain also a phosphatase acting on fructosamine 6-phosphates, which result from the reaction of proteins with glucose 6-phosphate. PMID:20967558

Van Schaftingen, Emile; Collard, François; Wiame, Elsa; Veiga-da-Cunha, Maria

2012-04-01

60

Regulation of Adherens Junction Dynamics by Phosphorylation Switches  

PubMed Central

Adherens junctions connect the actin cytoskeleton of neighboring cells through transmembrane cadherin receptors and a network of adaptor proteins. The interactions between these adaptors and cadherin as well as the activity of actin regulators localized to adherens junctions are tightly controlled to facilitate cell junction assembly or disassembly in response to changes in external or internal forces and/or signaling. Phosphorylation of tyrosine, serine, or threonine residues acts as a switch on the majority of adherens junction proteins, turning “on” or “off” their interactions with other proteins and/or their enzymatic activity. Here, we provide an overview of the kinases and phosphatases regulating phosphorylation of adherens junction proteins and bring examples of phosphorylation events leading to the assembly or disassembly of adherens junctions, highlighting the important role of phosphorylation switches in regulating their dynamics.

Bertocchi, Cristina; Vaman Rao, Megha; Zaidel-Bar, Ronen

2012-01-01

61

An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.  

PubMed

A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%). PMID:15121193

Hegeman, Adrian D; Harms, Amy C; Sussman, Michael R; Bunner, Anne E; Harper, Jeffrey F

2004-05-01

62

Enzymatic hydrolysis of molasses  

Microsoft Academic Search

Kinetic studies of the enzymatic hydrolysis of molasses were conducted using glucoamylase. Central Sugar Refinery SDN BHD contains 13–20% glucose. The molasses was diluted and the kinetic experiments were conducted at 67 °C with 100–1000 mg\\/l of glucoamylase. The glucose contents of the molasses were enhanced after hydrolysis of molasses solution with 1000 mg\\/l glucoamylase. A Lineweaver–Burk plot was obtained

Ghasem D. Najafpour; Cheong Poi Shan

2003-01-01

63

Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains  

SciTech Connect

High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 involving the nucleotide-binding (N) domain is required to permit the transfer of the -phosphoryl group of ATP to Asp351 in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientation of the N-domain relative to the P-domain in both different crystal structures of the Ca-ATPase following calcium activation, and structures of other P-type ATPases, suggests the presence of conformational heterogeneity in solution which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer (FRET) to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino] naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P- and N-domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains is unaffected by enzyme phosphorylation. However, calcium-activation results in a 3.4 increase in the average spatial separation, which increases from 29.4 to 32.8 , in good agreement with the high-resolution structures where these sites are respectively separated by 31.6 (1 IWO.pdb) and 35.9 (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. However, there is substantial conformational heterogeneity for all enzyme states measured, indicating that formation of catalytically important transition states involves a subpopulation of enzyme intermediates. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. These domain motions permit substrate (ATP) access and product (ADP) egress, and enhance the probability of a productive juxtaposition of the -phosphoryl moiety of ATP with Asp351 on the phosphosphorylation domain to facilitate enzyme phosphorylation and calcium transport.

Chen, Baowei; Squier, Thomas C.; Bigelow, Diana J.

2004-04-13

64

Bypassing AMPK Phosphorylation.  

PubMed

AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event and acts synergistically with AMP on naive AMPK. PMID:24856137

Viollet, Benoit; Foretz, Marc; Schlattner, Uwe

2014-05-22

65

Enzymatic cascade bioreactor  

DOEpatents

Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

Simmons, Blake A. (San Francisco, CA); Volponi, Joanne V. (Livermore, CA); Ingersoll, David (Albuquerque, NM); Walker, Andrew (Woodinville, WA)

2007-09-04

66

Phosphoryl-EZH-ion  

PubMed Central

Polycomb group (PcG) proteins regulate gene expression in embryonic and adult stem cells, but the mechanisms responsible for PcG gene targeting and regulation remain largely unknown. Recent evidence shows that EZH2, the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), is a nuclear phosphoprotein linking cell-cycle-intrinsic or extracellular signals to specific epigenetic signatures.

Caretti, Giuseppina; Palacios, Daniela; Sartorelli, Vittorio; Puri, Pier Lorenzo

2011-01-01

67

Phosphoryl-EZH-ion.  

PubMed

Polycomb group (PcG) proteins regulate gene expression in embryonic and adult stem cells, but the mechanisms responsible for PcG gene targeting and regulation remain largely unknown. Recent evidence shows that EZH2, the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), is a nuclear phosphoprotein linking cell-cycle-intrinsic or extracellular signals to specific epigenetic signatures. PMID:21362566

Caretti, Giuseppina; Palacios, Daniela; Sartorelli, Vittorio; Puri, Pier Lorenzo

2011-03-01

68

Struvite and prebiotic phosphorylation.  

NASA Technical Reports Server (NTRS)

Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

Handschuh, G. J.; Orgel, L. E.

1973-01-01

69

[Skeletal muscle troponin and phosphorylation: a site of troponin T, that is phosphorylated by specific protein kinase].  

PubMed

A procedure is described of the isolation of protein kinase, which phosphorylates isolated troponin T with a rate, 5--30 fold exceeding the phosphorylation rate of other substrates (phosvitine, caseine, protamine sulphate, H1, H2A, H2b, H3, H4 histones). Troponin T-specific protein kinase transfers 0.85--0.95 moles of P per 1 mol of dephosphorylated troponin T. It phosphorylates only N-terminal acetylated serine residue, i. e. the site of troponin T structure, which is normally phosphorylated, when the whole troponin complex is isolated from skeletal muscles. Protein kinase is incapable to phosphorylate N-terminal serine residue in a mixture of triptic peptides of troponon T. PMID:647084

Gusev, N B; Dobrovol'ski?, A B; Severin, S E

1978-02-01

70

Homogeneous, Heterogeneous, and Enzymatic Catalysis.  

ERIC Educational Resources Information Center

Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

Oyama, S. Ted; Somorjai, Gabor A.

1988-01-01

71

Economics of Enzymatic Hydrolysis Processes.  

National Technical Information Service (NTIS)

Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below tho...

J. D. Wright

1988-01-01

72

Enzymatic Template Polymerization, Part Two.  

National Technical Information Service (NTIS)

A conductive polymer is formed enzymatically in the presence of a polynucleotide template. The method includes combining at least one redox monomer with a polynucleotide template and a redox enzyme, such as horseradish peroxidase, to form a reaction mixtu...

L. A. Samuelson F. Bruno S. K. Tripathy S. Tripathy R. Nagarajan J. Kumar W. Liu

2004-01-01

73

Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum.  

PubMed

Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, ?-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases. PMID:24508535

Gao, Yanyan; Jiang, Ying; Liu, Qiulei; Wang, Ruiming; Liu, Xinli; Liu, Bo

2014-06-01

74

Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.  

PubMed Central

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.

Takahashi, N; Kalfas, S; Yamada, T

1995-01-01

75

Phosphorylated pyrone analogs and methods  

US Patent & Trademark Office Database

The invention relates to phosphorylated polyphenols, phosphorylated flavonoids, and phosphorylated pyrone analogs. Methods and compositions for the modulation of side effects of substances using such phosphorylated compounds are described. Methods and compositions are described for the modulation of blood-tissue barrier (BTB) transporter activity to increase the efflux of drugs and other compounds out of a physiological compartment and into an external environment. In particular, the methods and compositions disclosed herein provide lowered side effects when phosphorylated pyrone analogs are coadministered with therapeutic agents.

2011-05-24

76

Enzymatic Hydrolysis of Cellulosic Biomass  

SciTech Connect

Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

2011-08-22

77

Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis  

PubMed Central

The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation. DOI: http://dx.doi.org/10.7554/eLife.00691.001

Bolduc, David; Rahdar, Meghdad; Tu-Sekine, Becky; Sivakumaren, Sindhu Carmen; Raben, Daniel; Amzel, L Mario; Devreotes, Peter; Gabelli, Sandra B; Cole, Philip

2013-01-01

78

Molecular Evolution Allows Bypass of the Requirement for Activation Loop Phosphorylation of the Cdc28 Cyclin-Dependent Kinase  

Microsoft Academic Search

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phos- phorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone

FREDERICK R. CROSS; KRISTI LEVINE

1998-01-01

79

The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics**  

PubMed Central

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNASec) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNASec intermediate is converted into selenocysteinyl-tRNASec. The SepSecS architecture and the mode of tRNASec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNASec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNASec mimics that contain a hydrolysis-resistant ribose 3?-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNASec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNASec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes.

Rigger, Lukas; Schmidt, Rachel L.; Holman, Kaitlyn M.

2014-01-01

80

Blue light regulated two-component systems: enzymatic and functional analyses of light-oxygen-voltage (LOV)-histidine kinases and downstream response regulators.  

PubMed

Light is an essential environmental cue for diverse organisms. Many prokaryotic blue light photoreceptors use light, oxygen, voltage (LOV) sensory domains to control the activities of diverse output domains, including histidine kinases (HK). Upon activation, these proteins autophosphorylate a histidine residue before subsequently transferring the phosphate to an aspartate residue in the receiver domain of a cognate response regulator (RR). Such phosphorylation activates the output domain of the RR, leading to changes in gene expression, protein-protein interactions, or enzymatic activities. Here, we focus on one such light sensing LOV-HK from the marine bacterium Erythrobacter litoralis HTCC2594 (EL368), seeking to understand how kinase activity and subsequent downstream effects are regulated by light. We found that photoactivation of EL368 led to a significant enhancement in the incorporation of phosphate within the HK domain. Further enzymatic studies showed that the LOV domain affected both the LOV-HK turnover rate (kcat) and Km in a light-dependent manner. Using in vitro phosphotransfer profiling, we identified two target RRs for EL368 and two additional LOV-HKs (EL346 and EL362) encoded within the host genome. The two RRs include a PhyR-type transcriptional regulator (EL_PhyR) and a receiver-only protein (EL_LovR), reminiscent of stress-triggered systems in other bacteria. Taken together, our data provide a biochemical foundation for this light-regulated signaling module of sensors, effectors, and regulators that control bacterial responses to environmental conditions. PMID:23806044

Correa, Fernando; Ko, Wen-Huang; Ocasio, Victor; Bogomolni, Roberto A; Gardner, Kevin H

2013-07-01

81

Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.  

PubMed Central

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.

Dubiley, S; Kirillov, E; Lysov, Y; Mirzabekov, A

1997-01-01

82

Tyrosine phosphorylation of murine Crkl.  

PubMed

The SH2/SH3 adaptor protein Crkl is abnormally phosphorylated on tyrosine by the Bcr/Abl protein in leukemic cells from patients with Philadelphia-chromosome (Ph)positive leukemia. However, the state of tyrosine-phosphorylation of crkl in normal tissues is unknown. In the current study, we identified mouse crkl by cDNA cloning and examined expression levels and tyrosine-phosphorylation of the mouse crkl protein during embryogenesis and in adult tissues. Tyrosine-phosphorylation of crkl was prominent during early development, but decreased at later embryonic stages and in newborn mice. Expression of both crkl and the related crk was ubiquitous in the adult. However, crkl differed considerably from crk in relative tissue distribution, and was more abundant in hematopoietic tissues. With exception of the lung, crkl was mostly present in a non-tyrosine phosphorylated form. Consistent with our previous findings in human patients, murine crkl was phosphorylated on tyrosine in leukemic tissues of BCR/ABL transgenic animals, but was non-tyrosine phosphorylated in normal mouse bone marrow. We conclude that this crkl tyrosine-phosphorylation by Bcr/Abl in hematopoietic cells is clearly aberrant and is consistently linked to the development of leukemia. Identification of proteins interacting with tyrosine-phosphorylated crkl in the leukemic cells of BCR/ABL transgenic mice should reveal members of signal transduction pathways activated in Ph-positive leukemia. PMID:7478571

de Jong, R; Haataja, L; Voncken, J W; Heisterkamp, N; Groffen, J

1995-10-19

83

An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation*  

PubMed Central

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.

Turowec, Jacob P.; Zukowski, Stephanie A.; Knight, James D. R.; Smalley, David M.; Graves, Lee M.; Johnson, Gary L.; Li, Shawn S. C.; Lajoie, Gilles A.; Litchfield, David W.

2014-01-01

84

Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse*  

PubMed Central

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity.

Trinidad, Jonathan C.; Barkan, David T.; Gulledge, Brittany F.; Thalhammer, Agnes; Sali, Andrej; Schoepfer, Ralf; Burlingame, Alma L.

2012-01-01

85

Role of Mg ions in protein kinase phosphorylation: insights from molecular dynamics simulations of ATP-kinase complexes  

Microsoft Academic Search

Phosphorylation of protein kinases (PKs) plays a central role in the signal transduction of cells by transferring a phosphate group from adenosine triphosphate (ATP) to the side chains of particular serine, threonine or tyrosine residues of substrates. Previous studies have shown that Mg ions are crucial for the formation of transition state of phosphoryl transfer reaction. However, the roles of

Lu Yu; Lizhe Xu; Min Xu; Bo Wan; Long Yu; Qiang Huang

2011-01-01

86

Bioluminescence methods for enzymatic determinations  

SciTech Connect

An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

1982-11-02

87

Bioluminescence methods for enzymatic determinations  

DOEpatents

An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

Bostick, William D. (Oak Ridge, TN); Denton, Mark S. (Clinton, TN); Dinsmore, Stanley R. (Norris, TN)

1982-01-01

88

Improved enzymatic hydrolysis of hair.  

PubMed

An enzymatic hair extraction method is proposed for drug analysis. Pronase digestion of various aliquots of hair from a cocaine abuser was preceded by a 2-h incubation with a dithiothreitol solution. The extraction solution was tested to identify possible interferences in the radioimmunoassay and was compared with other hydrolysis methods to assess the results of extraction. PMID:8138218

Offidani, C; Strano Rossi, S; Chiarotti, M

1993-12-01

89

Phosphorylation-mediated inactivation of coactivator-associated arginine methyltransferase 1  

PubMed Central

Multiple protein arginine methyltransferases are involved in transcriptional activation of nuclear receptors. Coactivator-associated arginine methyltransferase 1 (CARM1)-mediated histone methylation has been shown to activate nuclear receptor-dependent transcription; however, little is known about the regulation of its enzymatic activity. Here, we report that the methyltransferase activity of CARM1 is negatively regulated through phosphorylation at a conserved serine residue. When the serine residue is mutated to glutamic acid, which mimics the phosphorylated serine residue, the mutant CARM1 exhibits diminished ability to bind the methyl donor adenosylmethionine and diminished histone methylation activity. Moreover, such mutation leads to the inhibition of CARM1 transactivation of estrogen receptor-dependent transcription. Our results provide an example for the regulation of protein arginine methyltransferase activity by phosphorylation. As CARM1 is a potent transcriptional coactivator of estrogen receptor, our results suggest that phosphorylation of CARM1 serves as a unique mechanism for inactivating CARM1-regulated estrogen-dependent gene expression.

Higashimoto, Ken; Kuhn, Peter; Desai, Dhaval; Cheng, Xiaodong; Xu, Wei

2007-01-01

90

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.  

PubMed

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action. PMID:10480872

Moeslein, F M; Myers, M P; Landreth, G E

1999-09-17

91

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells  

PubMed Central

Background Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. Results We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. Conclusion Widely-used adenovirus vectors for gene delivery cause a state of insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

Andersson, Monica; Warolen, Malin; Nilsson, Joakim; Selander, Martin; Sterky, Catharina; Bergdahl, Katrin; Sorving, Christina; James, Stephen R; Doverskog, Magnus

2007-01-01

92

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†  

PubMed Central

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

Deutscher, Josef; Francke, Christof; Postma, Pieter W.

2006-01-01

93

Phosphorescent sensor for phosphorylated peptides based on an iridium complex.  

PubMed

A bis[(4,6-difluorophenyl)pyridinato-N,C(2')]iridium(III) picolinate (FIrpic) derivative coupled with bis(Zn(2+)-dipicolylamine) (ZnDPA) was developed as a sensor (1) for phosphorylated peptides, which are related to many cellular mechanisms. As a control, a fluorescent sensor (2) based on anthracene coupled to ZnDPA was also prepared. When the total negative charge on the phosphorylated peptides was changed to -2, -4, and -6, the emission intensity of sensor 1 gradually increased by factors of up to 7, 11, and 16, respectively. In contrast, there was little change in the emission intensity of sensor 1 upon the addition of a neutral phosphorylated peptide, non-phosphorylated peptides, or various anions such as CO3(2-), NO3(-), SO4(2-), phosphate, azide, and pyrophosphate. Furthermore, sensor 1 could be used to visually discriminate between phosphorylated peptides and adenosine triphosphate in aqueous solution under a UV-vis lamp, unlike fluorescent sensor 2. This enhanced luminance of phosphorescent sensor 1 upon binding to a phosphorylated peptide is attributed to a reduction in the repulsion between the Zn(2+) ions due to the phenoxy anion, its strong metal-to-ligand charge transfer character, and a reduction in self-quenching. PMID:24919563

Kang, Jung Hyun; Kim, Hee Jin; Kwon, Tae-Hyuk; Hong, Jong-In

2014-07-01

94

Single-Molecule Enzymatic Dynamics  

SciTech Connect

Enzymatic turnovers of single cholesterol oxidase molecules are observed in real time by monitoring the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide (FAD). Although chemical kinetics, the Michaelis-Menten mechanism in particular, holds as a good approximation, statistical analyses of single-molecule trajectories reveal fluctuations in the rate of the activation step in the Michaelis-Menten mechanism. There exists a memory effect: an enzymatic turnover is not independent of its previous turnovers. This non-Markovian behavior, otherwise hidden in ensemble-averaged measurements, is attributed to slow fluctuations of protein conformations. Static heterogeneity and dynamical variation of reaction rates, essentially indistinguishable in ensemble-averaged experiments, can now be determined separately by the real-time single-molecule approach.

Lu, H. Peter; Xun, Luying; Xie, Xiaoliang

1998-12-04

95

The Enzymatic Oxidation of Graphene Oxide  

PubMed Central

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors.

Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

2011-01-01

96

Enzymatic hydrolysis of bacterial cellulose  

Microsoft Academic Search

Native cellulose from the bacterium Acetobacter xylinum as well as acid-treated bacterial cellulose prepared from partial hydrolysis of the native bacterial cellulose with 2.5 N HCl were subjected to enzymatic hydrolysis by Trichoderma viride cellobiohydrolase I (CBH I) and endoglucanase II (EG II). The activities of the two enzymes were continuously monitored with an oxidation-reduction potential electrode based on the

Masahiro Samejima; Junji Sugiyama; Kiyohiko Igarashi; Karl-Erik L. Eriksson

1997-01-01

97

Phosphorylation of Human Platelet Myosin  

PubMed Central

A preparation extracted from human blood platelets, which incorporates 32P from ?-labeled AT32P into one of the two light chains of platelet myosin and platelet myosin head is described. This phosphorylation, which appears to be due to an endogenous kinase, is specific for the myosin light chain in that no other protein extracted in 0.6 M KCl-15 mM Tris·HCl (pH 7.5) is phosphorylated. The phosphorylated light chain, which has been purified by gel filtration, releases the covalently bound phosphate after incubation in alkali and not after incubation in acid. Images

Adelstein, Robert S.; Conti, Mary Anne; Anderson, William

1973-01-01

98

Effects of alkoxy groups on arene rings of lignin ?-O-4 model compounds on the efficiencies of single electron transfer-promoted photochemical and enzymatic C-C Bond Cleavage Reactions.  

PubMed

To gain information about how alkoxy substitution in arene rings of ?-O-4 structural units within lignin governs the efficiencies/rates of radical cation C1-C2 bond cleavage reactions, single electron transfer (SET) photochemical and lignin peroxidase-catalyzed oxidation reactions of dimeric/tetrameric model compounds have been explored. The results show that the radical cations derived from less alkoxy-substituted dimeric ?-O-4 models undergo more rapid C1-C2 bond cleavage than those of more alkoxy-substituted analogues. These findings gained support from the results of DFT calculations, which demonstrate that C1-C2 bond dissociation energies of ?-O-4 radical cations decrease as the degree of alkoxy substitution decreases. In SET reactions of tetrameric compounds consisting of two ?-O-4 units, containing different degrees of alkoxy substitution, regioselective radical cation C-C bond cleavage was observed to occur in one case at the C1-C2 bond in the less alkoxy-substituted ?-O-4 moiety. However, regioselective C1-C2 cleavage in the more alkoxy-substituted ?-O-4 moiety was observed in another case, suggesting that other factors might participate in controlling this process. These observations show that lignins containing greater proportions of less rather than more alkoxylated rings as part of ?-O-4 units would be more efficiently cleaved by SET mechanisms. PMID:23992466

Lim, Suk Hyun; Nahm, Keepyung; Ra, Choon Sup; Cho, Dae Won; Yoon, Ung Chan; Latham, John A; Dunaway-Mariano, Debra; Mariano, Patrick S

2013-09-20

99

Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli.  

PubMed Central

It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI. We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII. In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI. NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction. Images

Weiss, V; Magasanik, B

1988-01-01

100

Phosphorylation and activation of the Jak-3 Janus kinase in response to interleukin-2  

Microsoft Academic Search

INTERLEUKIN-2 is an autocrine growth factor for T cells1,2 which also activates other cells including B cells3 and natural killer cells4. The subunits of the interleukin-2 receptor (IL-2R) lack intrinsic enzymatic activity, but protein tyrosine phosphorylation is a critical event following ligand binding and src family kinases, such as Lck, are known to be activated by IL-2 (refs 5-9). However,

James A. Johnston; Masaru Kawamura; Robert A. Kirken; Yi-Qing Chen; Trevor B. Blake; Kyoichi Shibuya; John R. Ortaldo; Daniel W. McVicar; John J. O'Shea

1994-01-01

101

Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays  

NASA Astrophysics Data System (ADS)

The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 ?m in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.

102

c-Myc phosphorylation by PKC? represses prostate tumorigenesis.  

PubMed

Studies showing reduced PKC? expression or enzymatic activity in different types of human cancers support the clinical relevance of PKC? as a tumor suppressor. However, the in vivo role of PKC? and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKC? in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKC?-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKC? knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKC? knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKC? is a critical event in the control of metastasis. Collectively, these results establish PKC? as an important tumor suppressor and regulator of c-Myc function in prostate cancer. PMID:23550155

Kim, Ji Young; Valencia, Tania; Abu-Baker, Shadi; Linares, Juan; Lee, Sang Jun; Yajima, Tomoko; Chen, Jing; Eroshkin, Alexey; Castilla, Elias A; Brill, Laurence M; Medvedovic, Mario; Leitges, Michael; Moscat, Jorge; Diaz-Meco, Maria T

2013-04-16

103

c-Myc phosphorylation by PKC? represses prostate tumorigenesis  

PubMed Central

Studies showing reduced PKC? expression or enzymatic activity in different types of human cancers support the clinical relevance of PKC? as a tumor suppressor. However, the in vivo role of PKC? and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKC? in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKC?-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKC? knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKC? knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKC? is a critical event in the control of metastasis. Collectively, these results establish PKC? as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

Kim, Ji Young; Valencia, Tania; Abu-Baker, Shadi; Linares, Juan; Lee, Sang Jun; Yajima, Tomoko; Chen, Jing; Eroshkin, Alexey; Castilla, Elias A.; Brill, Laurence M.; Medvedovic, Mario; Leitges, Michael; Moscat, Jorge; Diaz-Meco, Maria T.

2013-01-01

104

Heparanase augments EGF-receptor phosphorylation: correlation with head & neck tumor progression  

PubMed Central

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, a class of glycosaminoglycans abundantly present in the extracellular matrix and on the cell surface. Heparanase activity is strongly implicated in tumor metastasis attributed to remodeling of the subepithelial and subendothelial basement membranes resulting in dissemination of metastatic cancer cells. Moreover, heparanase up regulation was noted in an increasing number of primary human tumors, correlating with tumors larger in size, increased microvessel density, and reduced post operative survival rate, implying that heparanase function is not limited to tumor metastasis. This notion is supported by recent findings revealing induction of signaling molecules (i.e., Akt, p38) and gene transcription (i.e., tissue factor, VEGF) by enzymatically-inactive heparanase. Here, we provide evidence that active and inactive heparanase proteins enhance EGF-receptor (EGFR) phosphorylation. Enhanced EGFR phosphorylation was associated with increased cell migration, cell proliferation, and colony formation which were attenuated by Src inhibitors. Similarly, heparanase gene silencing by means of siRNA was associated with reduced Src and EGFR phosphorylation levels and decreased cell proliferation. Moreover, heparanase expression correlated with increased phospho-EGFR levels and progression of head and neck carcinoma, providing a strong clinical support for EGFR modulation by heparanase. Thus, heparanase appears to modulate two critical systems involved in tumor progression, namely VEGF expression and EGFR activation. Neutralizing heparanase enzymatic and non-enzymatic functions is therefore expected to profoundly affect tumor growth, angiogenesis, and metastasis.

Cohen-Kaplan, Victoria; Doweck, Ilana; Naroditsky, Inna; Vlodavsky, Israel; Ilan, Neta

2009-01-01

105

Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood.  

PubMed

The impact of oxidative modification and partial removal of lignin by laccase-mediator treatments on the enzymatic hydrolysis of steam-pretreated softwood (SPS) was evaluated. Two mediators, N-hydroxy-N-phenylacetamide (NHA) and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined. Both simultaneous and sequential combinations of laccase-mediator treatments with commercial cellulases increased the sugar yield in the enzymatic hydrolysis of SPS. The maximal increase was 21% when a sequential treatment was applied. Laccase treatment alone was also shown to improve hydrolysis. NHA oxidized by laccase inhibited significantly the cellulases of Trichoderma reesei, but the presence of the solid substrate protected the activities against oxidative inactivation. Surface analysis of the lignocellulosic substrate before and after the laccase and cellulase treatments revealed an enrichment of lignin and an increase of carboxylic groups on the surface of the hydrolysis residue. PMID:15129438

Palonen, Hetti; Viikari, Liisa

2004-06-01

106

Polymer synthesis by enzymatic catalysis.  

PubMed

Using biocatalysts as enzymes for in vitro polymer synthesis is a relatively new (third) stream that has become popular in the past two decades, following a first stream using catalysts of acids, bases, radical species, and so on, used since 1920s, and a second stream using catalysts of transition metals and rare metals since 1950s. Enzymatic catalysis is environmentally benign, yet allows the synthesis of various natural and unnatural polymers that have well-defined structures with controlled stereochemistry, regioselectivity, and chemoselectivity. This review will focus on recent developments in enzyme-catalyzed polymer synthesis in the areas of polysaccharides, polyesters, and polyaromatics, as well as polymer modification. PMID:20022287

Kadokawa, Jun-ichi; Kobayashi, Shiro

2010-04-01

107

Assay Development for the Determination of Phosphorylation Stoichiometry using MRM methods with and without Phosphatase Treatment: Application to Breast Cancer Signaling Pathways  

PubMed Central

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) detection with stable-isotope-labeled standard peptides. In contrast with the classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one non-phosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the % phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase, and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ER? and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites with important biological implications. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides, as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ER? protein revealed that the PPQ-MRM is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.

Domanski, Dominik; Murphy, Leigh C.; Borchers, Christoph H.

2010-01-01

108

Current Trends in Enzymatic Determination of Glycerol  

Microsoft Academic Search

This review focuses on the enzymatic glycerol detection methods. Importance of glycerol as biologically active compound, chemical glycerol determination methods, and current trends in enzymatic glycerol determination are reviewed. Application of FAD-dependent glycerol oxidases, NAD-dependent dehydrogenases, lipases and multi-enzymatic systems for glycerol and polyglyceride detection by optical and electrochemical methods are analyzed in detail. The application of PQQ-dependent enzymes is

I. Lapenaite; A. Ramanaviciene; A. Ramanavicius

2006-01-01

109

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization  

SciTech Connect

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

Geiss, Brian J. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Cano, Gina L. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Tavis, John E. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Morrison, Lynda A. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States)]. E-mail: morrisla@slu.edu

2004-12-05

110

Insulin-stimulated phosphorylation of recombinant pp120/HA4, an endogenous substrate of the insulin receptor tyrosine kinase.  

PubMed

Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 20,000. Analysis of the deduced amino acid sequence of rat liver pp120/HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr488 and Tyr513), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser503). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe488 and Ala503) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr488 is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser513 may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg(2+)-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg(2+)-dependent ecto-ATPase activity. PMID:7626603

Najjar, S M; Philippe, N; Suzuki, Y; Ignacio, G A; Formisano, P; Accili, D; Taylor, S I

1995-07-25

111

Multiple Peroxisomal Enzymatic Deficiency Disorders  

PubMed Central

Biologic, morphologic, and biochemical investigations performed in 2 patients demonstrate multiple peroxisomal deficiencies in the cerebrohepatorenal syndrome of Zellweger (CHRS) and neonatal adrenoleukodystrophy (NALD). Very long chain fatty acids, abnormal bile acids, including bile acid precursors (di- and trihydroxycoprostanoic acids), and C29-dicarboxylic acid accumulated in plasma in both patients. Generalized hyperaminoaciduria was also present. Peroxisomes could not be detected in CHRS liver and kidney; however, in the NALD patient, small and sparse cytoplasmic bodies resembling altered peroxisomes were found in hepatocytes. Hepatocellular and Kupffer cell lysosomes were engorged with ferritin and contained clefts and trilaminar structures believed to represent very long chain fatty acids. Enzymatic deficiencies reflected the peroxisomal defects. Hepatic glycolate oxidase and palmitoyl-CoA oxidase activities were deficient. No particle-bound catalase was found in cultured fibroblasts, and ether glycerolipid (plasmalogen) biosynthesis was markedly reduced. Administration of phenobarbital and clofibrate, an agent that induces peroxisomal proliferation and enzymatic activities, to the NALD patient did not bring about any changes in plasma metabolites, liver peroxisome population, or oxidizing activities. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5

Vamecq, Joseph; Draye, Jean-Pierre; Van Hoof, Francois; Misson, Jean-Paul; Evrard, Philippe; Verellen, Gaston; Eyssen, Hendrik J.; Van Eldere, Johan; Schutgens, Ruud B. H.; Wanders, Ronald J. A.; Roels, Frank; Goldfischer, Sidney L.

1986-01-01

112

Enzymatic formation of PEGylated oligonucleotides.  

PubMed

Gene therapy, siRNA, and therapeutic aptamers attract great interest owing to their versatility to treat a wide range of diseases and their potential high selectivity. Unfortunately, oligonucleotide-based therapeutics suffer rapid degradation by nucleases, scarce cell internalization, and fast kidney clearance. To address these limitations, the covalent attachment by mild chemical reactions of an activated polyethylene glycol (PEG) is widely used to obtain PEGylated nucleic acids showing a more favorable pharmacokinetic profile. We describe here a method for the enzymatic formation of PEGylated nucleic acids employing T4 DNA ligase: the ligation protocol was set up and optimized allowing the complete achievement of PEGylated oligonucleotides amenable to further enzymatic reactions. The feasibility of this approach for bioconjugation was demonstrated employing a set of PEG-donors and oligonucleotide acceptors, differing in the chemical link between PEG and the oligonucleotide donor, and in the length, sequence, and structure of the oligonucleotides employed. The ligase reaction allowed us to obtain double-stranded as well as single-stranded oligonucleotides, thus demonstrating the applicability of the method to a variety of substrates suitable for diagnostic and therapeutic applications. PMID:24450424

Sosic, Alice; Pasqualin, Matteo; Pasut, Gianfranco; Gatto, Barbara

2014-02-19

113

Method for the enzymatic production of hydrogen  

DOEpatents

The present invention is an enzymatic method for producing hydrogen comprising the steps of: a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch. The reaction mixture further comprises an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and c) detecting the hydrogen produced from the reaction mixture.

Woodward, Jonathan (Kingston, TN); Mattingly, Susan M. (State College, PA)

1999-01-01

114

Method for the enzymatic production of hydrogen  

DOEpatents

The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

Woodward, J.; Mattingly, S.M.

1999-08-24

115

Electrochemical non-enzymatic glucose sensors.  

PubMed

The electrochemical determination of glucose concentration without using enzyme is one of the dreams that many researchers have been trying to make come true. As new materials have been reported and more knowledge on detailed mechanism of glucose oxidation has been unveiled, the non-enzymatic glucose sensor keeps coming closer to practical applications. Recent reports strongly imply that this progress will be accelerated in 'nanoera'. This article reviews the history of unraveling the mechanism of direct electrochemical oxidation of glucose and making attempts to develop successful electrochemical glucose sensors. The electrochemical oxidation of glucose molecules involves complex processes of adsorption, electron transfer, and subsequent chemical rearrangement, which are combined with the surface reactions on the metal surfaces. The information about the direct oxidation of glucose on solid-state surfaces as well as new electrode materials will lead us to possible breakthroughs in designing the enzymeless glucose sensing devices that realize innovative and powerful detection. An example of those is to introduce nanoporous platinum as an electrode, on which glucose is oxidized electrochemically with remarkable sensitivity and selectivity. Better model of such glucose sensors is sought by summarizing and revisiting the previous reports on the electrochemistry of glucose itself and new electrode materials. PMID:17723330

Park, Sejin; Boo, Hankil; Chung, Taek Dong

2006-01-18

116

Interphase phosphorylation of lamin A.  

PubMed

Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

2014-06-15

117

The genesis of tyrosine phosphorylation.  

PubMed

Tyrosine phosphorylation of proteins was discovered in 1979, but this posttranslational modification had been "invented" by evolution more than a billion years ago in single-celled eukaryotic organisms that were the antecedents of the first multicellular animals. Because sophisticated cell-cell communication is a sine qua non for the existence of multicellular organisms, the development of cell-surface receptor systems that use tyrosine phosphorylation for transmembrane signal transduction and intracellular signaling seems likely to have been a crucial event in the evolution of metazoans. Like all types of protein phosphorylation, tyrosine phosphorylation serves to regulate proteins in multiple ways, including causing electrostatic repulsion and inducing allosteric transitions, but the most important function of phosphotyrosine (P.Tyr) is to serve as a docking site that promotes a specific interaction between a tyrosine phosphorylated protein and another protein that contains a P.Tyr-binding domain, such as an SH2 or PTB domain. Such docking interactions are essential for signal transduction downstream from receptor tyrosine kinases (RTKs) on the cell surface, which are activated on binding a cognate extracellular ligand, and, as a consequence, elicit specific cellular outcomes. PMID:24789824

Hunter, Tony

2014-01-01

118

Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components  

PubMed Central

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Ogura, Masato; Yamaki, Junko; Homma, Miwako K.; Homma, Yoshimi

2012-01-01

119

Nucleoside phosphorylation in amide solutions  

NASA Technical Reports Server (NTRS)

The paper deals with phosphorylation in possible prebiotic nonaqueous solvents. To this end, phosphorylation of nucleosides using inorganic phosphates in amide solutions is studied at room and elevated temperatures. Reaction proceeds most readily in formamide and N-methylformamide. Products obtained at elevated temperature are nucleotides, nucleoside 2',3'-cyclic phosphates, and when the phosphate concentration is high, nucleoside diphosphates. At room temperature, adenosine afforded a mixture of nucleotides, but none of the cyclic nucleotide. Conditions leading to the highest relative percentage of cyclic nucleotide involve the use of low concentrations of phosphate and an excess of nucleoside.

Schoffstall, A. M.; Kokko, B.

1978-01-01

120

The biochemistry and control of enzymatic browning  

Microsoft Academic Search

Half of the world's fruit and vegetable crops is lost due to postharvest deteriorative reactions. Polyphenol oxidase (PPO), found in most fruit and vegetables, is responsible for enzymatic browning of fresh horticultural products, following bruising, cutting or other damage to the cell. Chemical methods for controlling enzymatic browning include the use of sodium bisulfite, ascorbic acid and\\/or packaging under controlled

M. Victoria Martinez; John R. Whitaker

1995-01-01

121

Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.  

PubMed

An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity. PMID:24872080

Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

2014-06-12

122

Calcium ion-dependent phosphorylation of human erythrocyte membranes  

Microsoft Academic Search

Summary Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 µM. At nonlimiting ATP concentrations the level of32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane

Alcides F. Rega; Patricio J. Garrahan

1975-01-01

123

Enzymatic disease of the podocyte  

PubMed Central

Proteinuria is an early sign of kidney disease and has gained increasing attention over the past decade because of its close association with cardio-vascular and renal morbidity and mortality. Podocytes have emerged as the cell type that is critical in maintaining proper functioning of the kidney filter. A few genes have been identified that explain genetic glomerular failure and recent insights shed light on the pathogenesis of acquired proteinuric diseases. This review highlights the unique role of the cysteine protease cathepsin L as a regulatory rather than a digestive protease and its action on podocyte structure and function. We provide arguments why many glomerular diseases can be regarded as podocyte enzymatic disorders.

Kistler, Andreas D.; Peev, Vasil; Forst, Anna-Lena; El Hindi, Shafic; Altintas, Mehmet M.

2014-01-01

124

Stereochemical consequences of the use of chiral N-phosphoryl oxazolidinones in the attempted kinetic resolution of bromomagnesium alkoxides  

Microsoft Academic Search

A number of chiral N-phosphoryl oxazolidinones have been prepared and evaluated as asymmetric phosphoryl transfer agents with the magnesium alkoxide of 1-phenyl ethanol. The reaction proceeded with little stereoselection, which was shown to be a consequence of the reaction mechanism that occurs with inversion of configuration at phosphorus consistent with in-line attack opposite the leaving group.

Simon Jones; Dimitrios Selitsianos

2005-01-01

125

A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices.  

PubMed

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications. PMID:24333332

Spry, Christina; Saliba, Kevin J; Strauss, Erick

2014-04-15

126

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo¶  

PubMed Central

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown. Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309–382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.

Deterding, Leesa J.; Williams, Jason G.; Humble, Margaret M.; Petrovich, Robert M.; Wei, Sung-Jen; Trempus, Carol S.; Gates, Matthew B.; Zhu, Feng; Smart, Robert C.; Tennant, Raymond W.; Tomer, Kenneth B.

2010-01-01

127

SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION  

SciTech Connect

Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

JOHN C WALKER

2011-11-01

128

Phosphorylation in halobacterial signal transduction.  

PubMed Central

Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images

Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

1995-01-01

129

Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1  

PubMed Central

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1?AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta.

Scharein, Benjamin; Groth, Georg

2011-01-01

130

Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1.  

PubMed

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1-AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta. PMID:21912672

Scharein, Benjamin; Groth, Georg

2011-01-01

131

Stress-induced phosphorylation of SNAP-25.  

PubMed

Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser(187), however the physiological significance of this phosphorylation event in brain function remains unclear. In the present study, we found that SNAP-25 phosphorylation increased rapidly in the mouse brain following cold-water restraint stress. Both basal and stress-induced phosphorylation of SNAP-25 were high in stress-related brain regions, including the cerebral cortex, hippocampus, and amygdala, and the extent of phosphorylation increased with increasing amounts of stress. Intravenous administration of adrenaline increased SNAP-25 phosphorylation, although stress-induced phosphorylation was still observed in adrenalectomized mice. These results indicate that SNAP-25 phosphorylation is regulated in a stress-dependent manner through both central and peripheral mechanisms. PMID:24374286

Yamamori, Saori; Sugaya, Daichi; Iida, Yuuki; Kokubo, Hirotoshi; Itakura, Makoto; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

2014-02-21

132

Dephosphorylation of the Escherichia coli Transcriptional Antiterminator BglG by the Sugar Sensor BglF Is the Reversal of Its Phosphorylation  

PubMed Central

The Escherichia coli BglF protein catalyzes transport and phosphorylation of ?-glucosides. In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on ?-glucoside availability. Therefore, BglF has three enzymatic activities: ?-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG-P) dephosphorylase. Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG. To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF. Here we provide evidence which is consistent with the interpretation that Cys-24–P is an intermediate in the BglG-P dephosphorylation reaction. Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction.

Chen, Qing; Postma, Pieter W.; Amster-Choder, Orna

2000-01-01

133

Histidine to aspartate phosphotransferase activity of nm23 proteins: phosphorylation of aldolase C on Asp-319.  

PubMed Central

nm23 genes have been implicated in the suppression of tumour metastasis and cell motility; however, the biochemical mechanisms for these suppressions are not known. We have previously described the transfer of phosphate from the catalytic histidine residues of nm23 proteins to an aspartic or a glutamic residue on one or more 43 kDa proteins in detergent extracts of bovine brain membranes. To gain a better understanding of this transferase activity, we partly purified this 43 kDa protein and identified aldolases A and C as the major 43 kDa proteins present in the preparation. Aldolase was purified from brain cytosol; its phosphorylation by rat liver nm23 proteins and by recombinant human nm23-H1 was examined. The site of phosphorylation was identified as Asp-319 on aldolase C. The equivalent residue on aldolase A, a glutamic residue, was not phosphorylated. Aldolase C was rapidly phosphorylated by wild-type nm23-H1 but was not phosphorylated, or was phosphorylated very slowly, by either nm23-H1(P96S) or nm23-H1(S120G), mutants of nm23-H1 that do not suppress cell motility. This is the first identification of a protein that is phosphorylated on an aspartic residue by nm23 proteins. The sequence around Asp-319 of aldolase C has some similarities to those around the histidine residues on ATP-citrate lyase and succinic thiokinase that are phosphorylated by nm23 proteins.

Wagner, P D; Vu, N D

2000-01-01

134

Enzymatic degradation of thiolated chitosan.  

PubMed

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100?mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate-plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9-26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4-50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan. PMID:23057506

Laffleur, Flavia; Hintzen, Fabian; Rahmat, Deni; Shahnaz, Gul; Millotti, Gioconda; Bernkop-Schnürch, Andreas

2013-10-01

135

Enzymatic modification of flaxseed fibers.  

PubMed

Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers. PMID:23098092

Maijala, Pekka; Mäkinen, Marliina; Galkin, Sari; Fagerstedt, Kurt; Härkäsalmi, Tiina; Viikari, Liisa

2012-11-01

136

Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity  

PubMed Central

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor–bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain–containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2’s kinase and Shp2’s phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2–Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2.

Lin, Chi-Chuan; Suen, Kin M.; Melo, Fernando A.; Levitt, James A; Suhling, Klaus

2013-01-01

137

The semi-phosphorylative Entner-Doudoroff pathway in hyperthermophilic archaea: a re-evaluation  

PubMed Central

Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner–Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxygluconate) aldolase, has been cloned and characterized previously. In the present study, a comparative genomics analysis is described that reveals conserved ED gene clusters in both Thermoproteus tenax and S. solfataricus. The corresponding ED proteins from both archaea have been expressed in Escherichia coli and their specificity has been identified, revealing: (i) a novel type of gluconate dehydratase (gad gene), (ii) a bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate aldolase (kdgA gene), (iii) a 2-keto-3-deoxygluconate kinase (kdgK gene) and, in S. solfataricus, (iv) a GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; gapN gene). Extensive in vivo and in vitro enzymatic analyses indicate the operation of both the semi-phosphorylative and the non-phosphorylative ED pathway in T. tenax and S. solfataricus. The existence of this branched ED pathway is yet another example of the versatility and flexibility of the central carbohydrate metabolic pathways in the archaeal domain.

2005-01-01

138

Comprehensive Characterization of Heat Shock Protein 27 Phosphorylation in Human Endothelial Cells Stimulated by the Microbial Dithiole Thiolutin  

PubMed Central

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC–MS. Separate LC–MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC–MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC–MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC–MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.

Dai, Shujia; Jia, Yifeng; Wu, Shiaw-Lin; Isenberg, Jeff S.; Ridnour, Lisa A.; Bandle, Russell W.; Wink, David A.; Roberts, David D.; Karger, Barry L.

2009-01-01

139

Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin.  

PubMed

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones. PMID:18720982

Dai, Shujia; Jia, Yifeng; Wu, Shiaw-Lin; Isenberg, Jeff S; Ridnour, Lisa A; Bandle, Russell W; Wink, David A; Roberts, David D; Karger, Barry L

2008-10-01

140

Rapid Enzymatic Measurement of Serum Flucytosine Levels.  

National Technical Information Service (NTIS)

A method is provided for the rapid enzymatic determination of serum flucytosine or cytosine. The serum is first incubated with glutamate dehydrogenase to release endogenous ammonia which is measured, and the serum is then incubated with creatinine iminohy...

M. H. Kroll C. M. Huang R. G. Washburn

1987-01-01

141

IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY  

EPA Science Inventory

Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

142

Oxidative phosphorylation in Zymomonas mobilis  

Microsoft Academic Search

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles

U. Kalnenieks; A. A. Graaf; S. Bringer-Meyer; H. Sahm

1993-01-01

143

Oxidative phosphorylation in Micrococcus denitrificans  

Microsoft Academic Search

P\\/O ratios were measured in membrane particles obtained from cells of Micrococcus denitrificans, while growing on different carbon sources. The membrane particles obtained from cells growing actively on glucose, succinate, ethanol and propanol as the carbon and energy sources catalyzed oxidative phosphorylation and yielded respective P\\/O ratios of 1.4, 1.2, 0.8, and 0.5 with NADH, and 0.8, 0.6, 0.6, and

H. W. VAN VERSEVELD; A. H. Stouthamer

1976-01-01

144

Tyrosine phosphorylation in human lymphomas  

Microsoft Academic Search

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an\\u000a anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling\\u000a techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories\\u000a of lymphoma by staining 145 cases of lymphoma covering

E. Haralambieva; M. Jones; G. M. Roncador; L. Cerroni; L. Lamant; G. Ott; A. Rosenwald; C. Sherman; P. Thorner; R. Kusec; K. M. Wood; E. Campo; B. Falini; A. Ramsay; T. Marafioti; H. Stein; P. M. Kluin; K. Pulford; D. Y. Mason

2003-01-01

145

Specific regulatory role of phosphorylation of calf thymus DNA-topoisomerase I smaller forms on the relaxational activity expression. Phosphorylation role on Topo I smaller forms activity.  

PubMed

Calf thymus Topo I is found to be associated with three active breakdown products, resolved from intact enzyme, which do not appear to be unique to one extraction procedure. They are phosphoproteins, whose enzymatic activity can be modulated through changes in phosphorylation, and which can be phosphorylated 'in vitro', by N II protein kinase, in the same five sites as the intact enzyme. Different amounts of 32P incorporated are observed however, in the corresponding sites. We conclude: 1. proteolysis is probably an 'in vivo' phenomenon, as the Topo I smaller species are observed, during isolation from the earlier crude fractions, and as a minimum of them is always present, even if precautions are taken to minimize proteolysis; 2. a specific regulatory role in the DNA relaxational activity might be played by N II protein kinase phosphorylation, indeed, in the smaller species; 3. the different degrees of 32P incorporation, in analogous phosphorylation sites, might represent a different signal for modulating the gene expression. PMID:1318999

Coderoni, S; Paparelli, M; Gianfranceschi, G L

1992-05-01

146

Tyrosine phosphorylation and bacterial virulence  

PubMed Central

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.

Whitmore, Sarah E; Lamont, Richard J

2012-01-01

147

Stat1 Serine Phosphorylation Occurs Independently of Tyrosine Phosphorylation and Requires an Activated Jak2 Kinase  

Microsoft Academic Search

Gamma interferon (IFN-g) induces both tyrosine and serine phosphorylation of Stat1. Stat1 serine phos- phorylation is required for maximal transcriptional activity of Stat1. In this report, we present evidence that Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed

XUEJUN ZHU; ZILONG WEN; LIANG ZHONG XU; JAMES E. DARNELL

1997-01-01

148

Extracellular enzymatic activity of Malassezia spp. isolates  

Microsoft Academic Search

Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty\\u000a isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigatedusing Api Zym

Francesca Mancianti; Antonello Rum; Simona Nardoni; Michele Corazza

2001-01-01

149

Terahertz Biomems for Enzymatic Catalysis Monitoring  

Microsoft Academic Search

This work focuses on the fabrication and use of a Biological Micro-ElectroMechanical System (BioMEMS) for TeraHertz (THz) detection and characterization of enzymatic catalysis reactions. The fluidic microdevice was fabricated using traditional lithographic techniques. It was then functionalized by reactive amine functions using plasma polymerized allylamine (pp-allylamine), followed by enzyme immobilization inside the microchannels. The enzymatic reaction was controlled by fluorescent

Abdennour Abbas; Anthony Trezeibre; Nourredine Bourzgui; Didier Guillochon; Dominique Vercaigne-Marko; Philippe Supiot; Bertrand Bocquet

2009-01-01

150

Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase.  

PubMed

We have identified a novel mechanism of cross-talk between cell signaling and metabolic pathways, whereby the signaling kinase p21-activated kinase 1 (Pak1) binds to, phosphorylates and enhances the enzymatic activity of phosphoglucomutase 1 (PGM), an important regulatory enzyme in cellular glucose utilization and energy homeostasis. Pak1 and PGM were colocalized in model cell systems and showed functional interactions in a physiological setting. Strong direct interaction of PGM with Pak1 but not Pak2, Pak3, or Pak4 was observed. PGM binding was within 75-149 amino acids (aa) of Pak1, while Pak1 binding to PGM was in the N-terminal 96 aa. Pak1-mediated phosphorylation of PGM selectively on threonine 466 significantly increased PGM enzymatic activity and could be blocked by transfection with a dominant-negative Pak1 expression vector and by Pak1-specific small inhibitory RNA. Stable transfection of PGM into PGM-deficient K562 leukemia cells further demonstrated the role of Pak1 in regulating PGM activity. The results presented here provide new evidence that the cell signaling kinase Pak1 is a novel regulator of glucose metabolism through its phosphorylation and regulation of PGM activity. These findings suggest a new mechanism whereby growth factor signaling may coordinately integrate metabolic regulation with established signaling functions of cell cycle regulation and cell growth. PMID:15378030

Gururaj, Anupama; Barnes, Christopher J; Vadlamudi, Ratna K; Kumar, Rakesh

2004-10-21

151

Topoisomerase I tyrosine phosphorylation site and the DNA-interactive site  

SciTech Connect

Phosphorylation of topoisomerase I (topo I) at serine by NII kinase is accompanied by stimulation of enzymatic activity. In contrast, phosphorylation at tyrosine by tyrosine kinase seems to inhibit enzymatic activity. This inhibition may be caused by interference of the phosphorylated tyrosine residue with the interaction of topo I with DNA. To test this, topo I was labeled with crude membrane fraction enriched for EGF-receptor kinase in presence of ..gamma..-P32-ATP and electrophoresed on SDS-polyacrylamide gels. Stained topo I bands were excised, dried, digested with trypsin and analyzed on a C18 reverse-phase HPLC column. One major peak of radioactivity eluted at fraction 23 with 20% acetonitrile. To obtain the DNA-interactive site, topo I was incubated with pBR322 DNA labeled by nick-translation followed by DNase I treatment, and electrophoresis on SDS-polyacrylamide gels. Tryptic peptides were generated and analyzed by reverse-phase HPLC. A major peak of radioactivity eluted at fraction 16-18 with 15.5-17% acetonitrile. Studies are in progress to resolve whether (a) the two peptides are different, i.e. the tyrosine-P site and DNA-tyrosine interactive site are localized at different regions of the topo I or (b) the peptide sequences are identical but the covalent attachment of deoxynucleotides altered the peptide's elution from the HPLC column.

Roll, D.; Durban, E.

1986-05-01

152

Stimulations of Oxygen Uptake by Electron Transfer Inhibitors 1  

PubMed Central

The stimulation of oxygen uptake induced in avocado tissue slices by amytal, azide and cyanide has been studied. The effects of these inhibitors on O2 uptake and on phosphorylation suggest the coexistence of phosphorylating and non-phosphorylating electron transfer systems in the fruit. The reason for the stimulations of O2 uptake is believed to be the result of an increased supply of a limiting cofactor to the phosphorylating sites. The increased availability of cofactor per site is due to the inhibition of part of the cytochrome chain and the consequent reduction in the number of active phosphorylating sites.

Lips, S. Herman; Biale, Jacob B.

1966-01-01

153

Mycobacterium tuberculosis supports protein tyrosine phosphorylation.  

PubMed

Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ?1.4 million lives every year. PMID:24927537

Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S; Sherman, David R; Moritz, Robert L; Grundner, Christoph

2014-06-24

154

Bistability by Multiple Phosphorylation of Regulatory Proteins  

PubMed Central

The activity of a protein can be reversibly modulated by post-translational, covalent modifications, such as phosphorylation and dephosphorylation. In many cases, the modulated protein may be phosphorylated by the same kinase on many different amino acid residues. Such multisite phosphorylations may occur progressively (during a single binding event of kinase to substrate) or distributively (the kinase dissociates from its substrate after each phosphorylation reaction). If a protein is phosphorylated by a distributive multisite mechanism, then the net activity of a population of these protein molecules can be a highly nonlinear function of the ratio of activities of the kinase and phosphatase enzymes. If the multiply phosphorylated protein is embedded in a positive feedback loop with its kinase and/or phosphatase, then the network may exhibit robust bistable behavior. Using numerical simulations and bifurcation theory, we study the properties of a particular bistable reaction network motivated by the antagonistic relationship between cyclin-dependent kinase and its multiply phosphorylated target, Cdh1, which is involved in the degradation of cyclin molecules. We characterize the bistable switch in terms of (i) the mechanism of distributive phosphorylation (ordered or disordered), (ii) the number of phosphorylation sites on the target protein, (iii) the effect of phosphorylation on the target protein (abrupt or progressive inactivation), and (iv) the effects of stochastic fluctuations in small cells with limited numbers of kinase, phosphatase and target proteins.

Kapuy, Orsolya; Barik, Debashis; Domingo Sananes, Maria Rosa; Tyson, John J.

2009-01-01

155

Phosphorylation Regulates SIRT1 Function  

PubMed Central

Background SIR2 is an NAD+-dependent deacetylase [1]–[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo [9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. Methodology/Principal Findings We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. Conclusions/Significance Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]–[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions.

Sasaki, Tsutomu; Maier, Bernhard; Koclega, Katarzyna D.; Chruszcz, Maksymilian; Gluba, Wendy; Stukenberg, P. Todd; Minor, Wladek; Scrable, Heidi

2008-01-01

156

Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates.  

PubMed

We describe the synthesis of 2'-deoxyuridine-5'-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep Vent(R) exo(-) from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2'-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate. PMID:19707689

Borsenberger, Vinciane; Kukwikila, Mikiembo; Howorka, Stefan

2009-09-21

157

Enzymatic biotransformation of terpenes as bioactive agents.  

PubMed

The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3?,7?-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18? glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18? glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3?-hydroxy sclareol 18?-hydroxy sclareol, 6?,18?-dihydroxy sclareol, 11S,18?-dihydroxy sclareol, and 1?-hydroxy sclareol and 11S,18?-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1?-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases. PMID:23046385

Sultana, Nighat; Saify, Zafar Saeed

2013-12-01

158

Review: Enzymatic Hydrolysis of Cellulosic Biomass  

SciTech Connect

Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

2011-07-16

159

Use of enzymatic cleaners on US Navy ships. Research report  

SciTech Connect

The Naval Surface Warfare Center, Carderock Division, conducted a study to determine the feasibility of using enzymatic and bacterial products in cleaning applications aboard U.S. Navy ships. A review of the most recent technical literature and a survey of potential suppliers were conducted. In addition, shipboard systems, subsystems and housekeeping processes were evaluated to identify suitable applications for enzymatic and bacterial cleaners. The study identified numerous commercial products that, based on manufacturers` claims, would be effective and safe for use aboard ship to clean walls, floors, galley work surfaces, engine and machine parts, drains, pipes, grease traps, collection, holding and transfer (CHT) tanks, ballast tanks and bilge areas. However, the study also revealed the absence of standardized test protocols essential for validation of manufacturers` claims, and recommended the cooperative development of such protocols by representatives from the commercial sector, Government and academia. The need to obtain meaningful cost information based on actual use scenarios and to investigate any permitting issues associated with the discharge of related wastes to pierside facilities was also identified.

Venkatachalam, R.S.

1996-03-01

160

Prebiotic phosphorylation of nucleosides in formamide  

NASA Technical Reports Server (NTRS)

Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

Schoffstall, A. M.

1976-01-01

161

Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.  

PubMed

Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n?=?11), and in fibroblasts from healthy controls (n?=?11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines. PMID:23620374

De Paepe, Boel; Vandemeulebroecke, Katrien; Smet, Joél; Vanlander, Arnaud; Seneca, Sara; Lissens, Willy; Van Hove, Johan Lk; Deschepper, Ellen; Briones, Paz; Van Coster, Rudy

2014-02-01

162

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction, phosphorylation and ER localization.  

PubMed

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca(2+)-pumping. PMID:24727412

Ramírez Ríos, Sacnicte; Lamarche, Frédéric; Cottet-Rousselle, Cécile; Klaus, Anna; Tuerk, Roland; Thali, Ramon; Auchli, Yolanda; Brunisholz, René; Neumann, Dietbert; Barret, Luc; Tokarska-Schlattner, Malgorzata; Schlattner, Uwe

2014-08-01

163

The Geometry of Multisite Phosphorylation  

PubMed Central

Reversible protein phosphorylation on multiple sites is a key regulatory mechanism in most cellular processes. We consider here a kinase-phosphatase-substrate system with two sites, under mass-action kinetics, with no restrictions on the order of phosphorylation or dephosphorylation. We show that the concentrations of the four phosphoforms at steady state satisfy an algebraic formula—an invariant—that is independent of the other chemical species, such as free enzymes or enzyme-substrate complexes, and holds irrespective of the starting conditions and the total amounts of enzymes and substrate. Such invariants allow stringent quantitative predictions to be made without requiring any knowledge of site-specific parameter values. We introduce what we believe are novel methods from algebraic geometry—Gröbner bases, rational curves—to calculate invariants. These methods are particularly significant because they make it possible to treat parameters symbolically without having to specify their numerical values, and thereby allow us to sidestep the parameter problem. We anticipate that this approach will have much wider applications in biological modeling.

Manrai, Arjun Kumar; Gunawardena, Jeremy

2008-01-01

164

Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae.  

PubMed

Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily. PMID:11322729

Thompson, J; Robrish, S A; Pikis, A; Brust, A; Lichtenthaler, F W

2001-03-22

165

Structural Perspective on Enzymatic Halogenation  

PubMed Central

Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.; KannanR.; KopeckaH.; HarrisT. M.J. Am. Chem. Soc.1985, 42, 6652?6658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.; CobbS. L.; GeeA. D.; LockhartA.; MartarelloL.; McGlincheyR. P.; O’HaganD.; OnegaM.Chem. Commun.2006, 652?654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.; HuangF.; DengH.; SchaffrathC.; SpencerJ. B.; O’HaganD.; NaismithJ. H.Nature2004, 42, 561?56514765200). Structural characterization has provided a basis toward a mechanistic understanding of the specificity and chemistry of these enzymes. In particular, the latest crystallographic snapshots of active site architecture and halide binding sites have provided key insights into enzyme catalysis. Herein is a summary of the five classes of halogenases, focusing on the three most recently discovered: flavin-dependent halogenases, non-heme iron-dependent halogenases, and nucleophilic halogenases. Further, the potential roles of halide-binding sites in determining halide selectivity are discussed, as well as whether or not binding-site composition is always a seminal factor for selectivity. Expanding our understanding of the basic chemical principles that dictate the activity of the halogenases will advance both biology and chemistry. A thorough mechanistic analysis will elucidate the biological principles that dictate specificity, and the application of those principles to new synthetic techniques will expand the utility of halogenations in small-molecule development. PMID:14765200

2008-01-01

166

High consistency enzymatic hydrolysis of hardwood substrates.  

PubMed

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation. PMID:19643602

Zhang, Xiao; Qin, Wenjuan; Paice, Michael G; Saddler, John N

2009-12-01

167

Inactivation of Bcl-2 by phosphorylation.  

PubMed Central

The antiapoptosis potential of Bcl-2 protein is well established, but the mechanism of Bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid or the chemotherapeutic drug taxol, we found that Bcl-2 was phosphorylated in lymphoid cells. Phospho amino acid analysis revealed that Bcl-2 was phosphorylated on serine. Under similar conditions, okadaic acid or taxol treatment led to the induction of apoptosis in these cells. Thus, phosphorylation of Bcl-2 seems to inhibit its ability to interfere with apoptosis. In addition, phosphorylated Bcl-2 can no longer prevent lipid peroxidation as required to protect cells from apoptosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Haldar, S; Jena, N; Croce, C M

1995-01-01

168

eNOS phosphorylation on serine 1176 affects insulin sensitivity and adiposity  

PubMed Central

Phosphorylation of endothelial nitric oxide synthase (eNOS) is an important regulator of its enzymatic activity. We generated knockin mice expressing phosphomimetic (SD) and unphosphorylatable (SA) eNOS mutations at S1176 to study the role of eNOS phosphorylation. The single amino acid SA mutation is associated with hypertension and decreased vascular reactivity, while the SD mutation results in increased basal and stimulated endothelial NO production. In addition to these vascular effects, modulation of the S1176 phosphorylation site resulted in unanticipated effects on metabolism. The eNOS SA mutation results in insulin resistance, hyperinsulinemia, adiposity, and increased weight gain on high fat. In contrast, the eNOS SD mutation is associated with decreased insulin levels and resistance to high fat-induced weight gain. These results demonstrate the importance of eNOS in regulation of insulin sensitivity, energy metabolism, and bodyweight regulation, and suggest eNOS phosphorylation as a novel target for the treatment of obesity and insulin resistance.

Kashiwagi, Satoshi; Atochin, Dmitriy N.; Li, Qian; Schleicher, Michael; Pong, Terrence; Sessa, William C.; Huang, Paul L.

2013-01-01

169

Protein kinase C phosphorylation of PLC?1 regulates its cellular localization.  

PubMed

Activation of phospholipase C? (PLC?) by G proteins leads to a chain of events that result in an increase in intracellular calcium and activation of protein kinase C (PKC). It has been found that PKC phosphorylates PLC?1 on S887 in vitro without affecting its enzymatic activity or its ability to be activated by G?(q) proteins. To understand whether S887 phosphorylation affects the enzyme's activity in cells, we constructed two mutants that mimic the wild type and PKC-phosphorylated enzymes (S887A and S887D). We find that these constructs bind similarly to G?(q) in vitro. When expressed in HEK293 cells, both mutants associate identically to G?(q) in both the basal and stimulated states. Both mutants diffuse with similar rates and also interact identically with another known binding partner, translin-associated factor X (TRAX), which associates with PLC?1 in the cytosol and nucleus. However, the two mutants localize differently in the cell. We find that S887A has a much higher nuclear localization than its S887D counterpart both in HEK293 cells and PC12 cells. Our studies suggest that PKC phosphorylation regulates the level of PLC?1 cytosolic and nuclear activity by regulating its cellular compartmentalization. PMID:21338571

Aisiku, Omozuanvbo; Dowal, Louisa; Scarlata, Suzanne

2011-05-15

170

Extracellular Phosphorylation and Phosphorylated Proteins: Not Just Curiosities But Physiologically Important  

NSDL National Science Digital Library

With 3 figures, 1 table, and 221 references plus 2 supplementary figures and 1 supplementary table, this Review describes the evidence supporting physiological and pathological functions of phosphorylated extracellular proteins. In addition, evidence for extracellular phosphorylation in various contexts and tissues is also presented. To facilitate the study of the extracellular phosphoproteome, annotation of the phosphorylated proteins found in proteomic databases is supplied.

Garif Yalak (ETH Zurich;Department of Health Sciences and Technology REV); Viola Vogel (ETH Zurich;Department of Health Sciences and Technology REV)

2012-12-18

171

Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples.  

PubMed

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer. PMID:22191002

Paley, Elena L

2011-12-01

172

Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples1  

PubMed Central

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer.

Paley, Elena L

2011-01-01

173

Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome  

Microsoft Academic Search

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and

Matthias Mann; Shao-En Ong; Mads Grønborg; Hanno Steen; Ole N. Jensen; Akhilesh Pandey

2002-01-01

174

Protein tyrosine phosphorylation in cardiovascular system  

Microsoft Academic Search

Protein tyrosine phosphorylation is believed to play a central role in signaling pathways initiated by growth factor receptor activation. Recent studies have shown that various vasoactive peptides, in addition to eliciting a contractile response, also serve as growth factors for vascular smooth muscle and stimulate tyrosyl phosphorylation of several endogenous proteins. Some of these proteins have been identified and are

Ashok K. Srivastava

1995-01-01

175

Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains  

PubMed Central

Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc) translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB, and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB, and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding.

Toth, Stephen; Brueggmann, Ernst E.; Oyler, George A.; Smith, Leonard A.; Hines, Harry B.; Ahmed, S. Ashraf

2012-01-01

176

Systematic Discovery of In Vivo Phosphorylation Networks  

Microsoft Academic Search

SUMMARY Protein kinases control cellular decision pro- cesses by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome- wide mapping. However, systematically match- ing these sites to specific kinases is presently infeasible, due to limited specificity of consen- sus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on

Rune Linding; Lars Juhl Jensen; Gerard J. Ostheimer; Marcel A. T. M. van Vugt; Claus Jørgensen; Ioana M. Miron; Francesca Diella; Karen Colwill; Lorne Taylor; Kelly Elder; Pavel Metalnikov; Vivian Nguyen; Adrian Pasculescu; Jing Jin; Jin Gyoon Park; Leona D. Samson; James R. Woodgett; Peer Bork; Michael B. Yaffe; Tony Pawson

2007-01-01

177

Protein-tyrosine phosphorylation in the Archaea.  

PubMed Central

Sulfolobus sulfataricus ATCC 35091, Haloferax volcanii, and Methanosarcina thermophila TM-1, representing the Euryarchaeota and Crenarchaeota subdomains of the Archaea, contain proteins which are phosphorylated on tyrosine. These data raise fundamental questions as to the origin and evolution of tyrosine phosphorylation, a protein modification that is of pivotal importance in the regulation of the physiology of eukaryotic cells.

Smith, S C; Kennelly, P J; Potts, M

1997-01-01

178

pH & Rate of Enzymatic Reactions.  

ERIC Educational Resources Information Center

A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

Clariana, Roy B.

1991-01-01

179

Continuous Enzymatic Synthesis with Coenzyme Regeneration.  

National Technical Information Service (NTIS)

NAD(H) dependent enzymatic production of L-alanine and L-leucine was performed in an enzyme membrane reactor (EMR). An EMR is a continuous stirred tank reactor which is equipped with an ultrafiltration (UF) membrane at the exit of the reactor to retain en...

R. Wichmann

1981-01-01

180

Electrochemical non-enzymatic glucose sensors  

Microsoft Academic Search

The electrochemical determination of glucose concentration without using enzyme is one of the dreams that many researchers have been trying to make come true. As new materials have been reported and more knowledge on detailed mechanism of glucose oxidation has been unveiled, the non-enzymatic glucose sensor keeps coming closer to practical applications. Recent reports strongly imply that this progress will

Sejin Park; Hankil Boo; Taek Dong Chung

2006-01-01

181

Foaming properties of enzymatically hydrolysed wheat gluten  

Microsoft Academic Search

A thermally treated wheat gluten (TTG) was structurally modified by enzymatic hydrolysis. Hydrolysis was carried out using a fungal protease. The solubility of hydrolysates having low, medium and high degrees of hydrolysis (DH) and the foaming properties of their extracts at each pH were determined. For comparison, a sample of TTG was used as a standard. The results showed that

S. R. Drago; R. J. González

2000-01-01

182

Enzymatic saccharification of sugar-beet pulp  

Microsoft Academic Search

Nine commercial enzymatic preparations have been tested for their ability to release ferulic acid, rhamnose, arabinose, and galacturonic acid from sugar-beet pulp. SP 584, SP 585, and SP 342 from Novo Nordisk gave the highest release of ferulic acid, arabinose, and rhamnose. SP 584 and SP 585 degraded the pulp more rapidly than SP 342. Only SP 342 released free

V. Micard; C. M. G. C. Renard; J.-F. Thibault

1996-01-01

183

Enzymatic synthesis of amide surfactants from ethanolamine  

Microsoft Academic Search

The condensation of a primary amine with fatty acids has been studied to determine optimum conditions for selective formation of amide surfactants via enzymatic amidification. Monoacylated ethanolamide and the diacylated amide-ester can be isolated from the reaction mixture, but the monoacylated ester cannot be isolated. The selectivity of the reaction depends on the solubility of the intermediate amide. Continuous precipitation

Mónica Fernández-Pérez; Cristina Otero

2001-01-01

184

Heavy metal pollution and soil enzymatic activity  

Microsoft Academic Search

The activity of hydrolytic soil enzymes was studied on spruce mor, polluted with Cu and Zn from a brass foundry in Sweden. Approximately straight regression lines were obtained between enzymatic activity or respiration rate and log Cu+Zn concentration, with highly significant negative regression coefficients for urease and acid phosphatase activity as well as respiration rate, whereas ß-glucosidase activity was not

Germund Tyler

1974-01-01

185

Enzymatic processing of pheromones and pheromone analogs  

Microsoft Academic Search

Summary Pheromone perception requires rapid enzymatic degradation of the active chemical signal in the sensory hairs. Three insects are used to illustrate chemical approaches to studying the degradation of pheromones by antennal enzymes. First, hydrolysis of acetate and haloacetate esters is examined in the diamondback moth,Plutella xylostella. Second, aldehyde oxidation and the selective inhibition of the oxidase and dehydrogenase activities

G. D. Prestwich; S. McG Graham; M. Handley; B. Latli; L. Streinz; M. L. J. Tasayco

1989-01-01

186

Transfer form  

Cancer.gov

10/02 Transfer Investigational Agent Form This form is to be used for an intra-institutional transfer, one transfer/form. Division of Cancer Prevention National Cancer Institute National Institutes of Health TRANSFER FROM: Investigator transferring agent:

187

Manipulating and probing enzymatic conformational fluctuations and enzyme-substrate interactions by single-molecule FRET-magnetic tweezers microscopy.  

PubMed

Enzyme-substrate interaction plays a critical role in enzymatic reactions, forming the active enzyme-substrate complex, the transition state ready to react. Studying the enzyme-substrate interaction will help in the ultimate molecular-level characterization of the enzymatic transition state that defines the reaction pathway, energetics, and the dynamics. In our initial effort to experimentally investigate the enzyme-substrate interactions and the related conformational fluctuations, we have developed a new approach to manipulate the enzymatic conformation and enzyme-substrate interaction at a single-molecule level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule under the conditions with and without enzymatic substrates, we have probed and analyzed the enzymatic conformational dynamics. Our results indicate that the enzyme conformational flexibility can be regulated by enzyme-substrate interactions: (1) enzyme at its conformation-perturbed state has less flexibility when binding substrates, and (2) substrate binding to enzyme significantly changes the enzyme conformational flexibility, an experimental evidence of so called entropy trapping in the enzyme-substrate reactive transition state. Furthermore, our results provide a significant experimental analysis of the folding-binding enzyme-substrate interactions, a dynamic nature of the enzymatic active transition state formation process. PMID:24853252

Guo, Qing; He, Yufan; Lu, H Peter

2014-06-11

188

Quantitative analysis of aquaporin-2 phosphorylation  

PubMed Central

The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.

Xie, Luke; Hoffert, Jason D.; Chou, Chung-Lin; Yu, Ming-Jiun; Pisitkun, Trairak; Knepper, Mark A.

2010-01-01

189

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases.  

PubMed

This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions. PMID:11795479

Patel, M S; Korotchkina, L G

2001-12-31

190

Phosphorylation sites of protein kinase C ? in H2O2-treated cells and its activation by tyrosine kinase in vitro  

PubMed Central

Protein kinase C ? (PKC ?) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC ? in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC ? in response to H2O2.

Konishi, Hiroaki; Yamauchi, Emiko; Taniguchi, Hisaaki; Yamamoto, Toshiyoshi; Matsuzaki, Hidenori; Takemura, Yukitoshi; Ohmae, Kyoko; Kikkawa, Ushio; Nishizuka, Yasutomi

2001-01-01

191

The impact of N-terminal phosphorylation on LHCII conformation in state transition  

NASA Astrophysics Data System (ADS)

State transition is an important protection mechanism of plants for maintaining optimal efficiency through redistributing unbalanced excitation energy between photo-system II (PSII) and photosystem I (PSI). This process depends on the reversible phosphorylation/dephosphorylation of the major light-harvesting complex II (LHCII) and its bi-directional migration between PSII and PSI. But it remains unclear how phosphorylation/dephosphorylation modulates the LHCII conformation and further regulates its reversible migration. Here molecular dynamics simulations (MDS) were employed to elucidate the impact of phosphorylation on LHCII conformation. The results indicated that N-terminal phosphorylation loosened LHCII trimer with decreased hydrogen bond (H-bond) interactions and extended the distances between neighboring monomers, which stemmed from the conformational adjustment of each monomer itself. Global conformational change of LHCII monomer started from its stromal Nterminal (including the phosphorylation sites) by enhancing its interaction to lipid membrane and by adjusting the interaction network with surrounded inter-monomer and intra-monomer transmembrane helixes of B, C, and A, and finally triggered the reorientation of transmembrane helixes and transferred the conformational change to luminal side helixes and loops. These results further our understanding in molecular mechanism of LHCII migration during state transition from the phosphorylation-induced microstructural feature of LHCII.

Ding, Jin-Hong; Li, Ning; Wang, Man-Liu; Zhang, Yan; Lü, Shou-Qin; Long, Mian

2014-05-01

192

Enzymatic route to preparative-scale synthesis of UDP-GlcNAc/GalNAc, their analogues and GDP-fucose  

PubMed Central

Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP–GlcNAc/GalNAc. Other UDP–GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from l-fucose, GDP–fucose is constructed by one bifunctional enzyme l-fucose pyrophosphorylase (FKP) via two reactions.

Zhao, Guohui; Guan, Wanyi; Cai, Li; Wang, Peng George

2010-01-01

193

The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation.  

PubMed

Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages. PMID:16873379

Molle, Virginie; Brown, Alistair K; Besra, Gurdyal S; Cozzone, Alain J; Kremer, Laurent

2006-10-01

194

Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability*  

PubMed Central

Exotoxin Y (ExoY) is a type III secretion system effector found in ? 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.

Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

2012-01-01

195

The negative impact of ?-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation  

PubMed Central

A decline in ?-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ?30% higher ADP-ATP exchange rates compared to those obtained from DLST+/? or DLD+/? littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on “in-house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam-Vizi, V., Chinopoulos, C. The negative impact of ?-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation.

Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A.; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F.; Gibson, Gary E.; Beal, M. Flint; Adam-Vizi, Vera; Chinopoulos, Christos

2013-01-01

196

Src phosphorylation of cortactin enhances actin assembly  

PubMed Central

Src kinase mediates growth factor signaling and causes oncogenic transformation, which includes dramatic changes in the actin cytoskeleton, cell shape, and motility. Cortactin was discovered as a substrate for Src. How phosphorylation of cortactin can enhance actin assembly is unknown. Here, using an actin assembly system reconstituted from purified components, we demonstrate for the first time a biochemical mechanism by which Src phosphorylation of cortactin affects actin assembly. The adaptor Nck is an important component of the system, linking phosphorylated cortactin with neuronal WASp (N-WASp) and WASp-interacting protein (WIP) to activate Arp2/3 complex.

Tehrani, Shandiz; Tomasevic, Nenad; Weed, Scott; Sakowicz, Roman; Cooper, John A.

2007-01-01

197

Factors impeding enzymatic wheat gluten hydrolysis at high solid concentrations.  

PubMed

Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased maximum attainable degree of hydrolysis (DH%). We here identified the underlying factors causing the concentration effect. Wheat gluten was hydrolyzed at solid concentrations from 4.4% to 70%. The decreased hydrolysis rate was present at all solid concentrations and at any time of the reaction. Mass transfer limitations, enzyme inhibition and water activity were shown to not cause this hydrolysis rate limitation up to 50% solids. However, the hydrolysis rate limitation can be, at least partly, explained by a second-order enzyme inactivation process. Furthermore, mass transfer impeded the hydrolysis above 60% solids. Addition of enzyme after 24?h at high solid concentrations scarcely increased the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. Reduced enzyme activities caused by low water activities can explain this DH% limitation. Finally, a possible influence of the plastein reaction on the DH% limitation is discussed. Biotechnol. Bioeng. 2014;111: 1304-1312. © 2014 Wiley Periodicals, Inc. PMID:24474643

Hardt, N A; Janssen, A E M; Boom, R M; van der Goot, A J

2014-07-01

198

Haloperoxidases: Enzymatic Synthesis of ?,?-Halohydrins from Gaseous Alkenes  

PubMed Central

The enzymatic synthesis of ?,?-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from ?,?-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.

Geigert, John; Neidleman, Saul L.; Dalietos, Demetrios J.; DeWitt, Susanne K.

1983-01-01

199

Enzymatic treatment of sanitary landfill leachate.  

PubMed

The objective of this investigation was to study the effectiveness of applying enzymes (bioaugmentation) for enhancement of biological treatability of leachates generated in a typical municipal solid waste sanitary landfill. The basic purpose of enzyme use is to enforce the biodecomposition of organic constituents, as well as to reduce nitrogen content. A laboratory-scale sequencing batch (bio)reactor (SBR) was used for the examination of enzymatic application. The effect of different operation strategies on the efficiency of this biological treatment process was studied to optimize performance, especially for the removal of nitrogen compounds and of biodegradable organic matter. It was found that the enzymatic process was able to remove organic matter effectively (expressed as BOD5 and COD) and nitrogen content, color and turbidity. PMID:11513397

Zouboulis, A I; Loukidou, M X; Christodoulou, K

2001-08-01

200

A singular enzymatic megacomplex from Bacillus subtilis.  

PubMed

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs. PMID:17190806

Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

2007-01-01

201

Enzymatic catalysis of mercury methylation by Desulfovibrio desulfuricans LS.  

PubMed

The recently defined role of methylcobalamin in Hg2+ methylation by Desulfovibrio desulfuricans LS enabled us to reexamine the question of whether the principal source of methylmercury is spontaneous transmethylation or an enzymatically catalyzed process. In cell extracts of D. desulfuricans LS, over 95% of the 57Co label was associated with macromolecules rather than with free cobalamin. Both gel filtration and electrophoresis of cell extracts identified a single corrinoid protein of 40 kDa in size. This finding, in combination with the previously reported light-reversible propyl iodide inhibition of the Hg2+ methylation process, led us to propose that this 40-kDa corrinoid protein is the in vivo methyl donor in D. desulfuricans LS. Under reducing conditions, cell extracts containing the corrinoid protein produced 14CH3Hg+ from Hg2+ and 5-14CH3-tetrahydrofolate with a maximum specific activity of 0.73 nmol min-1 mg of cell protein-1. The sequence of methyl transfer was from methyltetrahydrofolate to the corrinoid protein to Hg2+. The rate of methylation versus the Hg2+ concentration followed Michaelis-Menten kinetics, with an apparent Km of 0.87 mM HgCl2. The activity was oxygen sensitive, and Hg2+ methylation was optimal at 35 degrees C and pH 6.5. The observation of saturation kinetics and the 600-fold-higher rate of Hg2+ methylation (at pH 7.0) by cell extracts, compared with transmethylation by free methylcobalamin, proved that in vivo Hg2+ methylation is an enzymatically catalyzed process. PMID:8017921

Choi, S C; Chase, T; Bartha, R

1994-04-01

202

Enzymatic catalysis of mercury methylation by Desulfovibrio desulfuricans LS.  

PubMed Central

The recently defined role of methylcobalamin in Hg2+ methylation by Desulfovibrio desulfuricans LS enabled us to reexamine the question of whether the principal source of methylmercury is spontaneous transmethylation or an enzymatically catalyzed process. In cell extracts of D. desulfuricans LS, over 95% of the 57Co label was associated with macromolecules rather than with free cobalamin. Both gel filtration and electrophoresis of cell extracts identified a single corrinoid protein of 40 kDa in size. This finding, in combination with the previously reported light-reversible propyl iodide inhibition of the Hg2+ methylation process, led us to propose that this 40-kDa corrinoid protein is the in vivo methyl donor in D. desulfuricans LS. Under reducing conditions, cell extracts containing the corrinoid protein produced 14CH3Hg+ from Hg2+ and 5-14CH3-tetrahydrofolate with a maximum specific activity of 0.73 nmol min-1 mg of cell protein-1. The sequence of methyl transfer was from methyltetrahydrofolate to the corrinoid protein to Hg2+. The rate of methylation versus the Hg2+ concentration followed Michaelis-Menten kinetics, with an apparent Km of 0.87 mM HgCl2. The activity was oxygen sensitive, and Hg2+ methylation was optimal at 35 degrees C and pH 6.5. The observation of saturation kinetics and the 600-fold-higher rate of Hg2+ methylation (at pH 7.0) by cell extracts, compared with transmethylation by free methylcobalamin, proved that in vivo Hg2+ methylation is an enzymatically catalyzed process.

Choi, S C; Chase, T; Bartha, R

1994-01-01

203

Total enzymatic synthesis of cholecystokinin CCK5  

Microsoft Academic Search

Summary. This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH 2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt·HCl

H. Xiang; G. Y. Xiang; Z. M. Lu; L. Guo; H. Eckstein

2004-01-01

204

Enzymatic acylglycerol synthesis in a membrane bioreactor  

Microsoft Academic Search

Lipases can catalyze the esterification reaction in a two-phase system. TheCandida rugosa lipase-catalyzed esterification of decanoic acid with glycerol is described in this work for an emulsion system and for a\\u000a hydrophilic membrane bioreactor. The enzymatic activity is studied in relation to the interface area between the two phases,\\u000a the enzyme load and the reactor volume. The initial rate per

A. van der Padt; M. J. Edema; J. J. W. Sewalt; K. van't Riet

1990-01-01

205

Molecular crowding and protein enzymatic dynamics.  

PubMed

The effects of molecular crowding on the enzymatic conformational dynamics and transport properties of adenylate kinase are investigated. This tridomain protein undergoes large scale hinge motions in the course of its enzymatic cycle and serves as prototype for the study of crowding effects on the cyclic conformational dynamics of proteins. The study is carried out at a mesoscopic level where both the protein and the solvent in which it is dissolved are treated in a coarse grained fashion. The amino acid residues in the protein are represented by a network of beads and the solvent dynamics is described by multiparticle collision dynamics that includes effects due to hydrodynamic interactions. The system is crowded by a stationary random array of hard spherical objects. Protein enzymatic dynamics is investigated as a function of the obstacle volume fraction and size. In addition, for comparison, results are presented for a modification of the dynamics that suppresses hydrodynamic interactions. Consistent with expectations, simulations of the dynamics show that the protein prefers a closed conformation for high volume fractions. This effect becomes more pronounced as the obstacle radius decreases for a given volume fraction since the average void size in the obstacle array is smaller for smaller radii. At high volume fractions for small obstacle radii, the average enzymatic cycle time and characteristic times of internal conformational motions of the protein deviate substantially from their values in solution or in systems with small density of obstacles. The transport properties of the protein are strongly affected by molecular crowding. Diffusive motion adopts a subdiffusive character and the effective diffusion coefficients can change by more than an order of magnitude. The orientational relaxation time of the protein is also significantly altered by crowding. PMID:22476233

Echeverria, Carlos; Kapral, Raymond

2012-04-01

206

TERAHERTZ BIOMEMS FOR ENZYMATIC CATALYSIS MONITORIN G  

Microsoft Academic Search

This work focuses on the fabrication and use of a B iological Micro-ElectroMechanical System (BioMEMS) for TeraHertz (THz) detection and characterization of e nzymatic catalysis reactions. The fluidic microdevi ce was fabricated using traditional lithographic techniques. It was t hen functionalized by reactive amine functions usin g plasma polymerized allylamine (pp-allylamine), followed by enzyme immobilization inside the microchannels. The enzymatic

Abdennour Abbas; Anthony Trezeibre; Nourredine Bourzgui; Didier Guillochon; Dominique Vercaigne-Marko; Philippe Supiot; Bertrand Bocquet

2009-01-01

207

Enzymatic transformation of nonfood biomass to starch.  

PubMed

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y-H Percival

2013-04-30

208

Enzymatic transformation of nonfood biomass to starch  

PubMed Central

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

2013-01-01

209

Protein tyrosine phosphorylation in cardiovascular system.  

PubMed

Protein tyrosine phosphorylation is believed to play a central role in signaling pathways initiated by growth factor receptor activation. Recent studies have shown that various vasoactive peptides, in addition to eliciting a contractile response, also serve as growth factors for vascular smooth muscle ans stimulate tyrosyl phosphorylation of several endogenous proteins. Some of these proteins have been identified and are similar to those stimulated by growth factor receptor activation. Furthermore, evidence is also accumulating to support an involvement of protein tyrosine phosphorylation in acute action of growth factors and vasoactive peptides on smooth an muscle contractility. This review still briefly summarize the recent work on vasoactive peptide-mediated protein tyrosine phosphorylation in cardiovascular tissues and its potential functional significance. PMID:8569753

Srivastava, A K

1995-01-01

210

Tyrosine phosphorylation in human lymphomas.  

PubMed

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL. PMID:14626344

Haralambieva, E; Jones, M; Roncador, G M; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, K M; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, P M; Pulford, K; Mason, D Y

2002-01-01

211

Enzymatic hydrolysis of penicillin for 6-APA production in three-liquid-phase system.  

PubMed

A dodecane/thermosensitive polymer/water three-liquid-phase system was introduced for enzymatic hydrolysis of penicillin G (Pen G) for 6-aminopenicillanic acid (6-APA). The enzyme was covalently attached to the terminal of PEO-PPO-PEO polymer (L63), which would be transferred into a polymer coacervate phase at high temperature above its "cloud point". 6-APA was primarily resided in the aqueous phase due to its zwitterionic nature. More than 70% phenylacetic acid (PAA) was transferred into the organic phase using trioctylmethylammonium hydroxide and trihexyl-(tetradecyl)phosphonium bis 2,4,4-trimethylpentylphosphinate ionic liquids (Cyphos IL-104) mixture at pH 5.5, while most of Pen G resided in water. As a result, high operational pH was permitted in three-liquid-phase system, which leads to higher enzymatic activity (120 IU at 40 degrees C) and stability (enzymatic half-time up to 55 h at 60 degrees C) in comparison with the value in butyl acetate/water two-phase system. On the other hand, two products in three-liquid-phase system might be automatically separated from the enzyme sphere into different phases at the same time, which facilitated the reaction equilibrium towards the product's side with 6-APA productivity of 80% at 42 degrees C, pH 5.5. PMID:18456946

Jiang, Yangyang; Xia, Hansong; Guo, Chen; Mahmood, Iram; Liu, Huizhou

2008-02-01

212

Prediction of Cyclin-Dependent Kinase Phosphorylation  

Microsoft Academic Search

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28

Substrates Emmanuel; J. Chang; Rashida Begum; Brian T. Chait; Terry Gaasterland

213

Cold enzymatic bleaching of fluid whey.  

PubMed

Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically bleached WPC80, lactoperoxidase-bleached WPC80 contained higher relative abundance of 2,3-octadienone, 2-pentyl furan, and hexanal than those bleached with added EP. Bleach times, bleaching efficacy, and flavor results suggest that enzymatic bleaching may be a viable and desirable alternative to HP bleaching of fluid whey or retentate. PMID:24140314

Campbell, R E; Drake, M A

2013-12-01

214

Protein phosphorylation in neurodegeneration: friend or foe?  

PubMed Central

Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system.

Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

2014-01-01

215

Protein phosphorylation: Localization in regenerating optic axons  

SciTech Connect

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

Larrivee, D. (Cornell Univ. Medical College, New York, NY (USA))

1990-09-01

216

Protein Kinase C Phosphorylation of PLC?1 Regulates its Cellular Localization  

PubMed Central

Activation of phospholipase C? (PLC?) by G proteins leads to a chain of events that result in an increase in intracellular calcium and activation of protein kinase C (PKC). It has been found that PKC phosphorylates PLC?1 on S887 in vitro without affecting its enzymatic activity or its ability to be activated by G?(q) proteins. To understand whether S887 phosphorylation affects the enzyme's activity in cells, we constructed two mutants that mimic the wild type and PKC-phosphoryated enzymes (S887A and S887D). We find that these constructs bind similarly to G?(q) in vitro. When expressed in HEK293 cells, both mutants associate identically to G?(q) in both the basal and stimulated states. Both mutants diffuse with similar rates and also interact identically with another known binding partner, Translin-Associated factor X (TRAX), which associates with PLC?1 in the cytosol and nucleus. However, the two mutants localize differently in the cell. We find that S887A has a much higher nuclear localization than its S887D counterpart both in HEK293 cells and PC12 cells. Our studies suggest that PKC phosphorylation regulates the level of PLC?1 cytosolic and nuclear activity by regulating its cellular compartmentalization.

Aisiku, Omozuanvbo; Dowal, Louisa; Scarlata, Suzanne

2011-01-01

217

Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status  

PubMed Central

Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4 h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization.

Ladurner, Angela; Schmitt, Christoph A.; Schachner, Daniel; Atanasov, Atanas G.; Werner, Ernst R.; Dirsch, Verena M.; Heiss, Elke H.

2012-01-01

218

Fragmentation of phosphorylated and singly charged peptide ions via interaction with metastable atoms.  

PubMed

Fragmentation of phosphorylated peptide ions via interaction with electronically excited metastable argon atoms was studied in a linear trap - time-of-flight mass spectrometer. Doubly charged ions of phosphorylated peptides from an Enolase digest were produced by electrospray ionization and subjected to a metastable atom beam in the linear trap. The metastable argon atoms were generated using a glow-discharge source. An intensive series of c- and z- ions were observed in all cases, with the phosphorylation group intact. The formation of molecular radical cations with reduced charge indicated that an electron transfer from a highly excited metastable state of argon to the peptide cation occurred. Additionally, singly charged Bradykinin, Substance P and Fibrinopeptide A molecular ions were fragmented via interaction with electronically excited metastable helium atoms. The fragmentation mechanism was different in this case and involved Penning ionization. PMID:19956340

Berkout, Vadym D; Doroshenko, Vladimir M

2008-12-01

219

Fragmentation of phosphorylated and singly charged peptide ions via interaction with metastable atoms  

PubMed Central

Fragmentation of phosphorylated peptide ions via interaction with electronically excited metastable argon atoms was studied in a linear trap – time-of-flight mass spectrometer. Doubly charged ions of phosphorylated peptides from an Enolase digest were produced by electrospray ionization and subjected to a metastable atom beam in the linear trap. The metastable argon atoms were generated using a glow-discharge source. An intensive series of c- and z- ions were observed in all cases, with the phosphorylation group intact. The formation of molecular radical cations with reduced charge indicated that an electron transfer from a highly excited metastable state of argon to the peptide cation occurred. Additionally, singly charged Bradykinin, Substance P and Fibrinopeptide A molecular ions were fragmented via interaction with electronically excited metastable helium atoms. The fragmentation mechanism was different in this case and involved Penning ionization.

Berkout, Vadym D.; Doroshenko, Vladimir M.

2008-01-01

220

Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed  

PubMed Central

Background Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed. Results Changes in glucan hydrolysis and PSD were measured as a function of agitation during enzymatic hydrolysis at fiber loadings of 7 or 13% water-insoluble solids (WIS). Enzymatic conversion of pretreated spruce was strongly affected by agitation rates at the higher WIS content. However, at low WIS content the agitation had almost no effect on hydrolysis. There was some effect of agitation on the hydrolysis of giant reed at high WIS loading, but it was smaller than that for spruce, and there was no measurable effect at low WIS loading. In the case of spruce, intense agitation clearly affected the PSD and resulted in a reduced mean particle size, whereas for giant reed the decrease in particle size was mainly driven by enzymatic action. However, the rate of enzymatic hydrolysis was not increased after size reduction by agitation. Conclusions The impact of agitation on the enzymatic hydrolysis clearly depends not only on feedstock but also on the solids loading. Agitation was found to affect the PSD differently for the examined pretreated materials spruce and giant reed. The fact that the reduced mean particle diameter could not explain the enhanced hydrolysis rates found for spruce at an elevated agitation suggests that mass transfer at sustained high viscosities plays an important role in determining the rate of enzymatic hydrolysis.

2014-01-01

221

Structural dynamics of muscle protein phosphorylation.  

PubMed

We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain, the striated muscle accessory protein myosin binding protein-C, and the cardiac membrane Ca(2+) pump modulator phospholamban. In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany. PMID:22930331

Colson, Brett A; Gruber, Simon J; Thomas, David D

2012-12-01

222

Structural dynamics of muscle protein phosphorylation  

PubMed Central

We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain (RLC), the striated muscle accessory protein myosin binding protein-C (MyBP-C), and the cardiac membrane Ca2+ pump modulator phospholamban (PLB). In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.

Colson, Brett A.; Gruber, Simon J.; Thomas, David D.

2013-01-01

223

Phosphorylated lymphocyte plasma-membrane proteins.  

PubMed Central

Lymphocytes were labelled by incubation with [32P]Pi and their plasma membranes isolated. Analysis by one-dimensional and two-dimensional gel electrophoresis revealed a small number of strongly phosphorylated polypeptides. Two of these were especially prominent; they had molecular weights of about 52000 and 90000, were acidic and were apparently not glycosylated. Similar patterns were obtained for quiescent T- and B-lymphocytes from different species and for cultured lymphoblastoid cells, although the relative amounts of the labelled polypeptides varied. Immunoprecipitation analyses of the detergent-solubilized 32P-labelled plasma membranes indicated that the glycosylated polypeptide of the human major transplantation (HLA-A and HLA-B) antigens and its mouse and pig counterparts are phosphorylated. In contrast, no phosphorylation of the membrane-associated immunoglobulin, the mouse Thy-1 antigen or the human HLA-DRw(Ia) antigen was detected. The phosphorylation patterns of human peripheral blood and nude-mouse spleen lymphocytes did not change during the period 5-30min after mitogen stimulation. Therefore a change in the phosphorylation of plasma-membrane protein(s) is probably not an early biochemical event in the initiation of T-lymphocyte and B-lymphocyte growth, although a rapid transient change cannot be ruled out. Similar plasma-membrane phosphorylation patterns were also obtained by incubating the purified plasma membrane with [gamma-32P]ATP. The phosphorylation of the 90000-mol.wt. polypeptide was particularly rapid and was stimulated by the addition of cyclic AMP. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6.

Johnstone, A P; DuBois, J H; Crumpton, M J

1981-01-01

224

Studying the fragmentation behavior of peptides with arginine phosphorylation and its influence on phospho-site localization.  

PubMed

Phospho-proteomic studies opened a broad view onto the main mechanisms of regulating cellular processes. Our recent discovery of a protein arginine kinase and its target in bacteria added a previously undescribed type of phosphorylation to control protein activity. Several challenges arise from large in vivo studies of this and other types of phosphorylations. The main factors impeding correct localization are low spectral quality, neutral loss of phosphoric acid, and gas-phase rearrangements, which have recently been described for phospho-serine, -threonine, and -tyrosine. Studies on histidine-phosphorylated peptides, a nitrogen-bound phosphorylation, also reported loss of phosphoric acid upon collision-induced dissociation. We were interested in studying the behaviour of arginine phosphorylation under different fragmentation conditions and its influence on site localization. First, we determined the percentage of false localizations obtained by three different search engines and a software tool dedicated for phospho-site determination. Next, we demonstrate that application of collisional activation for analysis of arginine-phosphorylated peptides leads to extensive elimination of phosphoric acid and increases the numbers of false localizations, while the modification is maintained on the arginine side chain upon electron-transfer dissociation. Furthermore, we also observed a rearrangement of the phosphorylation onto serine and glutamic acid side chains upon collisional activation. PMID:23172725

Schmidt, Andreas; Ammerer, Gustav; Mechtler, Karl

2013-03-01

225

Mutations affecting enzymatic activity in liver arginase  

SciTech Connect

The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

Vockley, J.G.; Tabor, D.E.; Goodman, B.K. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

1994-09-01

226

Kinetic behaviour of liver glucokinase in diabetes. II. Possible role of non-enzymatic protein glycation.  

PubMed

The phosphorylation of D-glucose, as catalyzed by liver postmicrosomal supernatants, prepared from diabetic rats, under conditions aiming at the characterization of gluco-kinase activity, indicates, in addition to the classical fall in enzyme activity, an altered kinetic behaviour, the affinity for D-glucose and the apparent energy of activation being both lower in diabetic than normal rats. These kinetic anomalies persist after separation of cytosolic proteins from low molecular weight metabolites by gel filtration chromatography. They are simulated, to a limited extent, when liver cytosolic proteins from normal rats are glycated in vitro through prolonged exposure to a high concentration of D-glucose. Diabetes causes an increased non-enzymatic glycation of liver cytosolic proteins, including lactate dehydrogenase, as judged by either the ketoamine test, a back-titration procedure or the separation of glycated proteins by affinity chromatography. These findings suggest that chronic hyperglycemia might alter the intrinsic properties of liver glucokinase through a process of non-enzymatic glycation. PMID:2132181

Zähner, D; Ramirez, R; Malaisse, W J

1990-07-01

227

Enzymatic saccharification of defatted corn germ.  

PubMed

Commercial defatted germ from wet milled corn was efficiently saccharified by a crude enzyme preparation from Aureobasidium sp. with yields of up to 200 mg glucose, 140 mg xylose, and 130 mg arabinose per g germ. These yields exceeded sugar composition estimates based on trifluoroacetic acid digestion. Neither chemical nor mechanical pretreatments were necessary. Results from independent lots of defatted germ were similar. Enzymatically digested germ residues were enriched to 40% (w/v) protein. Defatted germ from dry milled corn contained approx. 50% more starch than wet milled germ and was saccharified to produce up to 315 mg glucose per g germ with reduced yields of pentose sugars. PMID:15049363

Leathers, Timothy D

2004-02-01

228

Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1.  

PubMed

Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1. PMID:22320399

Veisova, Dana; Macakova, Eva; Rezabkova, Lenka; Sulc, Miroslav; Vacha, Petr; Sychrova, Hana; Obsil, Tomas; Obsilova, Veronika

2012-05-01

229

Phosphorylation of Lipin 1 and Charge on the Phosphatidic Acid Head Group Control Its Phosphatidic Acid Phosphatase Activity and Membrane Association*  

PubMed Central

The lipin gene family encodes a class of Mg2+-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

Eaton, James M.; Mullins, Garrett R.; Brindley, David N.; Harris, Thurl E.

2013-01-01

230

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF).  

PubMed

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling. PMID:19146952

Daviau, Alex; Di Fruscio, Marco; Blouin, Richard

2009-04-01

231

Systematic Discovery of In Vivo Phosphorylation Networks  

PubMed Central

Summary Protein kinases control cellular decision processes by phosphorylating specific substrates. Proteome-wide mapping has identified thousands of in vivo phosphorylation sites. However, systematically resolving which kinase targets each site is presently infeasible, due to the limited specificity of consensus motifs and the potential influence of contextual factors, such as protein scaffolds, localisation and expression, on cellular substrate specificity. We have therefore developed a computational method, NetworKIN, that augments motifs with context for kinases and phosphoproteins. This can pinpoint individual kinases responsible for specific in vivo phosphorylation events and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. We show that context provides 60–80% of the computational capability to assign in vivo substrate specificity. Applying this approach to a DNA damage signalling network, we extend its cell-cycle regulation by showing that 53BP1 is a CDK1 substrate, show that Rad50 is phosphorylated by ATM kinase under genotoxic stress, and suggest novel roles of ATM in apoptosis. Finally, we present a scalable strategy to validate our predictions and use it to support the prediction that BCLAF1 is a GSK3 substrate.

Linding, Rune; Jensen, Lars Juhl; Ostheimer, Gerard J.; van Vugt, Marcel A.T.M.; J?rgensen, Claus; Miron, Ioana M.; Diella, Francesca; Colwill, Karen; Taylor, Lorne; Elder, Kelly; Metalnikov, Pavel; Nguyen, Vivian; Pasculescu, Adrian; Jin, Jing; Park, Jin Gyoon; Samson, Leona D.; Woodgett, James R.; Russell, Robert B.; Bork, Peer; Yaffe, Michael B.; Pawson, Tony

2009-01-01

232

Processing and phosphorylation of the Fat receptor  

PubMed Central

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. We show here that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. Our observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance our understanding of the requirement for Dco in Fat signaling.

Feng, Yongqiang; Irvine, Kenneth D.

2009-01-01

233

Protein phosphorylation systems in postmortem human brain  

SciTech Connect

Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. (Rockefeller Univ., New York, NY (USA))

1989-01-01

234

Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.  

PubMed Central

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

D'Oro, U; Sakaguchi, K; Appella, E; Ashwell, J D

1996-01-01

235

Enzymatic and bacterial conversions during sourdough fermentation.  

PubMed

Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds. PMID:24230468

Gänzle, Michael G

2014-02-01

236

Mapping the Reaction Coordinates of Enzymatic Defluorination  

PubMed Central

The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights of how this formidable feat is achieved. Here, we present a series of high-resolution (1.15–1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion, but is also finely tailored for the smaller fluorine halogen atom to establish selectivity towards fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling and regulatory strategies for the growing markets of organofluorines across major industrial sectors.

Chan, Peter W. Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.

2011-01-01

237

Palm date fibers: analysis and enzymatic hydrolysis.  

PubMed

Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J

2010-01-01

238

Categorization of some oscillatory enzymatic reactions  

SciTech Connect

We investigate the categorization of two or more proposed reaction mechanisms for each of the following oscillatory enzymatic reactions: (1) the peroxidase-oxidase reaction; (2) glycolytic oscillations; (3) oscillations of cyclic AMP in smile mold cells; (4) enzymatic pH oscillations; (5) calcium spiking in cytosol. We use prior work in stoichiometric network analysis and categorization of oscillatory reactions to identify in each proposed reaction mechanism essential and nonessential species, the specific role of each essential species, the connectivity of the essential species, including the identification of the reactions leading to oscillatory instabilities, and the category. For each model, we predict the result of several experiments including relative amplitudes, quench amplitudes, phase shifts, and sign symbolic concentration shifts and compare them with those from available experiments. These and several other experiments such as bifurcation analysis, phase response curves, entrainment experiments, qualitative and quantitative pulsed species response, delay experiments, and external periodic perturbation provide stringent tests of proposed reaction mechanisms, and appropriate ones are suggested to discriminate among competing mechanisms for a given reaction. We find the necessity for introducing a new subcategory in our categorization of oscillatory reactions. 45 refs., 6 figs., 6 tabs.

Schreiber, I. [Prague Inst. of Chemical Technology (Czechoslovakia)] [Prague Inst. of Chemical Technology (Czechoslovakia); Hung, Y.F.; Ross, J. [Stanford Univ., CA (United States)] [Stanford Univ., CA (United States)

1996-05-16

239

Probing enzymatic activity inside single cells.  

PubMed

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 ?M ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells. PMID:24003961

Olofsson, Jessica; Xu, Shijun; Jeffries, Gavin D M; Jesorka, Aldo; Bridle, Helen; Isaksson, Ida; Weber, Stephen G; Orwar, Owe

2013-11-01

240

Enzymatic deconstruction of xylan for biofuel production  

PubMed Central

The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction.

DODD, DYLAN; CANN, ISAAC K. O.

2010-01-01

241

Enzymatic deconstruction of xylan for biofuel production.  

PubMed

The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO(2)) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO(2) into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716

Dodd, Dylan; Cann, Isaac K O

2009-02-18

242

Palm Date Fibers: Analysis and Enzymatic Hydrolysis  

PubMed Central

Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes.

Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J.

2010-01-01

243

Allergenic potential and enzymatic resistance of buckwheat  

PubMed Central

Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy.

Lee, Sujin; Han, Youngshin; Do, Jeong-Ryong

2013-01-01

244

Allergenic potential and enzymatic resistance of buckwheat.  

PubMed

Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy. PMID:23423876

Lee, Sujin; Han, Youngshin; Do, Jeong-Ryong; Oh, Sangsuk

2013-02-01

245

Phosphorylation Reaction in cAPK Protein Kinase - Free Energy Quantum Mechanic/Molecular Mechanics Simulations.  

SciTech Connect

Protein kinases catalyze the transfer of the ?-phosphoryl group from ATP, a key regulatory process governing signalling pathways in eukaryotic cells. The structure of the active site in these enzymes is highly conserved implying common catalytic mechanism. In this work we investigate the reaction process in cAPK protein kinase (PKA) using a combined quantum mechanics and molecular mechanics approach. The novel computational features of our work include reaction pathway determination with nudged elastic band methodology and calculation of free energy profiles of the reaction process taking into account finite temperature fluctuations of the protein environment. We find that the transfer of the ?-phosphoryl group in the protein environment is an exothermic reaction with the reaction barrier of 15 kcal/mol.

Valiev, Marat; Yang, Jie; Adams, Joseph; Taylor, Susan S.; Weare, John H.

2007-11-29

246

Enzymatic quantification of sphingosine in the picomole range in cultured cells  

SciTech Connect

An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-(32P)sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.

Van Veldhoven, P.P.; Bishop, W.R.; Bell, R.M. (Duke Univ. Medical Center, Durham, NC (USA))

1989-11-15

247

Focus Issue: Systems Analysis of Protein Phosphorylation  

NSDL National Science Digital Library

Kinases and phosphatases are key regulatory molecules that participate in most cell signaling pathways. Systems-level analyses are providing new insights into phosphorylation sites and kinase specificity, and phosphoproteomic analyses are creating not just a wealth of data, but are also revealing rich revelations about cellular behavior, cellular responses to changing environmental conditions, and mechanisms of disease. Phosphatases are not merely signal terminators, but play active roles in signal transduction and are especially important in redox signaling. In this Focus Issue, Science Signaling highlights protein phosphorylation as the center of the signaling universe.

Nancy R. Gough (American Association for the Advancement of Science;Science Signaling REV); John F. Foley (American Association for the Advancement of Science;Science Signaling REV)

2010-08-31

248

Deregulation of Cytoskeletal Protein Phosphorylation and Neurodegeneration  

Microsoft Academic Search

\\u000a Phosphorylation of cytoskeletal proteins is tightly regulated by the activities of multiple protein kinases and phosphatases.\\u000a Three kinases, Cyclin-dependent kinase 5 (Cdk5), Glycogen synthase kinase 3? (GSK3?) and MAPKs have been implicated in their\\u000a direct involvement in neuronal cytoskeletal protein phosphorylation. Cdk5 and GSK3? have been identified as prime candidates\\u000a for pathogenesis. Cdk5 is a proline-directed serine\\/threonine protein kinase that

Jyotshnabala Kanungo; Ya-li Zheng; Parvathi Rudrabhatla; Niranjana D. Amin; Bibhutibhushan Mishra; Harish C. Pant

249

Positive Correlation between Tyrosine Phosphorylation of CpsD and Capsular Polysaccharide Production in Streptococcus pneumoniae  

Microsoft Academic Search

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modu- lation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell

Matthew H. Bender; Robert T. Cartee; Janet Yother

2003-01-01

250

SPONTANEOUS AND CATALYZED HYDROGEN SHIFTS IN RADICAL CATIONS HAVING A PHOSPHORYL OR CARBONYL GROUP  

Microsoft Academic Search

Intermolecular and intramolecular hydrogen shifts represent a key component of a vast number of chemical reactions. This is particularly true for radical cations, whose high reactivity makes them prone to isomerization and dissociation reactions. In the context of the experimental work in this thesis, hydrogen transfers involved in both the intra-and inter-molecular isomerization of radical cations containing a phosphoryl (P=O)

Lisa Natasha Heydorn

2003-01-01

251

Kinetics of PCr to ATP and ?-ATP to ?-ADP phosphoryl conversion are modified in working rat skeletal muscle after training  

Microsoft Academic Search

Kinetics of phosphoryl transfers from PCr to ?-ATP and from ?-ATP to ?-ADP were measured by magnetization transfer in an in\\u000a vivo31P NMR experiment in working rat skeletal hind leg muscles. Two groups were examined. One group was submitted to a 6-week training\\u000a program of treadmill running. The other group was composed of sedentary animals. Metabolic oxidative capacity and mechanical

Xavier Ravalec; Nathalie Le Tallec; François Carré; Jacques D. de Certaines; Elisabeth Le Rumeur

1999-01-01

252

Sensitive Electrochemical Detection of Enzymatically-generated Thiocholine at Carbon Nanotube Modified Glassy Carbon Electrode  

SciTech Connect

A carbon nanotube modified glassy-carbon (CNT/GC) electrode was used for enhancing the sensitivity of electrochemical measurements of enzymatically-generated thiocholine. Cyclic voltammetric and amperometric characteristics of thiocholine at CNT/GC, glassy carbon, carbon paste, and gold electrodes were compared. The CNT layer leads to a greatly improved anodic detection of enzymatically generated thiocholine product including lower oxidation overpotential (0.15 V) and higher sensitivity because of its electrocatalytic activity, fast electron transfer and large surface area. The sensor performance was optimized with respect to the operating conditions. Under the optimal batch conditions, a detection limit of 5 ?10 -6 mol/L was obtained with good precision (RSD = 5.2%, n=10). Furthermore, the attractive response of thiocholine on a CNT/GC electrode has allowed it to be used for constant-potential flow injection analysis. The detection limit was greatly improved to 0.3 ?10-6 mol/L. The high sensitivity electrochemical detection of enzymatically generated thiocholine with a CNT sensing platform holds great promise to prepare an acetylcholinesterase biosensor for monitoring organophosphate pesticides and nerve agents.

Liu, Guodong; Riechers, Shawn L.; Mellen, Maria C.; Lin, Yuehe

2005-11-01

253

Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues  

PubMed Central

Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization.

1995-01-01

254

Enzymatic hydrolysis of rawhide using papain and neutrase  

Microsoft Academic Search

Rawhide split was hydrolysed separately by two proteolytic enzymes, papain and neutrase. The effects of enzymatic conditions of the hydrolysis reaction were investigated. During the first 10min of the enzymatic hydrolysis, the yield of the hydrolysed protein increased sharply, then it slowly increased or became essentially constant due to the limited availability of the substrate. The optimum hydrolysis conditions of

Siriporn Damrongsakkul; Kongpob Ratanathammapan; Kittinan Komolpis; Wiwut Tanthapanichakoon

2008-01-01

255

An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides  

Microsoft Academic Search

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and

Gustav Vaaje-Kolstad; Bjørge Westereng; Svein J. Horn; Zhanliang Liu; Hong Zhai; Morten Sørlie; Vincent G. H. Eijsink

2010-01-01

256

Article Requirement for an Enzymatic Visual Cycle in Drosophila  

Microsoft Academic Search

Summary Background: The visual cycle is an enzymatic pathway employed in the vertebrate retina to regenerate the chromo- phore after its release from light-activated rhodopsin. How- ever, a visual cycle is thought to be absent in invertebrates such as the fruit fly Drosophila melanogaster. Results: We demonstrate that an enzymatic visual cycle exists in flies for chromophore regeneration and requires

Xiaoyue Wang; Tao Wang; Yuchen Jiao; Johannes von Lintig; Craig Montell

257

Use of enzymatic cleaners on US Navy ships. Research report  

Microsoft Academic Search

The Naval Surface Warfare Center, Carderock Division, conducted a study to determine the feasibility of using enzymatic and bacterial products in cleaning applications aboard U.S. Navy ships. A review of the most recent technical literature and a survey of potential suppliers were conducted. In addition, shipboard systems, subsystems and housekeeping processes were evaluated to identify suitable applications for enzymatic and

Venkatachalam

1996-01-01

258

Physicochemical characterisation of enzymatically hydrolysed derivatives of acetylated starch  

Microsoft Academic Search

Potato starch modified to different degrees by substitution with acetyl groups was the subject of this study undertaken to determine the influence of conditions of enzymatic hydrolysis on the surface-active properties of hydrolysates of acetylated starch. The effect of acetylation of starch preparation on its susceptibility to enzymatic hydrolysis in the membrane reactor was also considered. All hydrolysates of acetylated

Emilia Konowa?; Gra?yna Lewandowicz; Joanna Le Thanh-Blicharz; Krystyna Prochaska

259

ENHANCING ENZYMATIC HYDROLYSIS OF RICE STRAW BY MICROWAVE PRETREATMENT  

Microsoft Academic Search

In an attempt to elucidate the effect of microwave pretreatment (MP) on enzymatic hydrolysis of rice straw (RS), three distinct MP procedures, microwave treatment (MT) alone, MT and alkali treatment (AT) together, and MT after AT, were investigated and compared with conventional alkali pretreatment (AP) by measurement of reducing sugar during its enzymatic hydrolysis. The combination of MT and AT

Zhu Shengdong; Yu Ziniu; Wu Yuanxin; Zhang Xia; Li Hui; Gao Ming

2005-01-01

260

Synthesis of optically active strigolactones: enzymatic resolution and asymmetric hydroxylation  

Microsoft Academic Search

A synthetic intermediate of an optically active strigolactone analogue was prepared in two ways: enzymatic resolution and asymmetric hydroxylation. The 4-hydroxy tricyclic lactone 4 was enzymatically resolved to give the corresponding enantiomers in an enantiomerically pure state, while the tricyclic lactone 5 was hydroxylated asymmetrically at the 4-position by the action of cytochrome P450 monooxygenase.

Hirosato Takikawa; Hiromasa Imaishi; Aya Tanaka; Satoshi Jikumaru; Mami Fujiwara; Mitsuru Sasaki

2010-01-01

261

Ion channels, phosphorylation and mammalian sperm capacitation  

PubMed Central

Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

Visconti, Pablo E; Krapf, Dario; de la Vega-Beltran, Jose Luis; Acevedo, Juan Jose; Darszon, Alberto

2011-01-01

262

Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia.  

PubMed

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia. PMID:7521685

ten Hoeve, J; Arlinghaus, R B; Guo, J Q; Heisterkamp, N; Groffen, J

1994-09-15

263

Dynamics of protein phosphorylation during meiotic maturation  

PubMed Central

Purpose To ask whether distinct kinase signaling pathways mediate cytoplasmic or nuclear maturation of mouse oocytes and if in vitro maturation influences the distribution and timing of these phosphorylation events. Methods Mouse cumulus oocyte complexes (COCs) were matured under conditions known to influence oocyte quality (basal or supplemented media) and assayed with epitope specific antibodies that would distinguish between Cdk1 or tyrosine kinase targets at 0, 2, 4, 8, and 16 hrs. Semi-quantitative image analysis was used to assess the topographical patterns of protein phosphorylation during in vitro maturation. In vitro fertization and embryo culture were used to examine the effects of culture conditions on developmental potential. Results Protein tyrosine phosphorylation increased during meiotic progression from methaphase-I to metaphase-II. Levels were significantly higher in the oocyte cortex. Levels of cortical staining are enhanced in oocytes matured in supplemented media that displayed higher developmental competence. In contrast, bulk substrates for Cdk1 kinase localize to the meiotic spindle while cytoplasmic levels of kinase activity increase throughout meiotic progression; culture media had no measurable effect. Ablation of the tyrosine kinase Fyn significantly reduced cortical levels of tyrosine phosphorylation. Conclusions The findings indicate that distinct signaling pathways mediate nuclear and cytoplasmic maturation during in vitro maturation in a fashion consistent with a role for tyrosine kinases in cortical maturation and oocyte quality.

Albertini, David F.

2010-01-01

264

The genetics and pathology of oxidative phosphorylation  

Microsoft Academic Search

The mitochondrial oxidative phosphorylation (OXPHOS) system is the final biochemical pathway in the production of ATP. The OXPHOS system consists of five multiprotein complexes, the individual subunits of which are encoded either by the mitochondrial or by the nuclear genome. Defects in the OXPHOS system result in devastating, mainly multisystem, diseases, and recent years have seen the description of the

Jan Smeitink; Lambert van den Heuvel; Salvatore DiMauro

2001-01-01

265

APP processing is regulated by cytoplasmic phosphorylation  

Microsoft Academic Search

myloid- ? peptide (A ? ) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers

Ming-Sum Lee; Shih-Chu Kao; Cynthia A. Lemere; Weiming Xia; Huang-Chun Tseng; Ying Zhou; Rachael Neve; Michael K. Ahlijanian; Li-Huei Tsai

2003-01-01

266

Regulation of the CFTR channel by phosphorylation  

Microsoft Academic Search

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles

David Dahan; Alexandra Evagelidis; John W. Hanrahan; Deborah A. R. Hinkson; Yanlin Jia; Jiexin Luo; Tang Zhu

2001-01-01

267

Colors as catalysts in enzymatic reactions.  

PubMed

We studied the effects of visible range irradiation (in vitro) on the enzyme solutions (glucose oxidase, cholesterol oxidase + cholesterol esterase and lipase) in order to infer the changes produced in the human body after chromotherapy. The glucose oxidase showed enhanced activity to the color purple (464 nm), while the activity of the other enzymes, cholesterol esterase + cholesterol oxidase and lipase, increased when exposed to dark violet (400 nm). Purple is being used in conventional chromotherapy for diabetes, as supported by the experimental observation in which purple enhanced the activity of enzymes responsible for the oxidation of glucose. Specific wavelengths regulate living processes by acting as catalysts in enzyme activity, while some wavelengths may reduce enzyme activity. The irradiation of specific wavelengths effect enzymatic processes, which as a consequence, accelerated biochemical reactions. This particular frequency when provided to the enzymes (in vitro) lead to changes which may well be occurring in vivo. PMID:20633466

Azeemi, Samina T Yousuf; Raza, Syed Mohsin; Yasinzai, Masoom

2008-12-01

268

Zinc oxide inverse opal enzymatic biosensor  

NASA Astrophysics Data System (ADS)

We report ZnO inverse opal- and nanowire (NW)-based enzymatic glucose biosensors with extended linear detection ranges. The ZnO inverse opal sensors have 0.01-18 mM linear detection range, which is 2.5 times greater than that of ZnO NW sensors and 1.5 times greater than that of other reported ZnO sensors. This larger range is because of reduced glucose diffusivity through the inverse opal geometry. The ZnO inverse opal sensors have an average sensitivity of 22.5 ?A/(mM cm2), which diminished by 10% after 35 days, are more stable than ZnO NW sensors whose sensitivity decreased by 10% after 7 days.

You, Xueqiu; Pikul, James H.; King, William P.; Pak, James J.

2013-06-01

269

Thermal stability of bioactive enzymatic papers.  

PubMed

The thermal stability of two enzymes adsorbed on paper, alkaline phosphatase (ALP) and horseradish peroxidase (HRP), was measured using a colorimetric technique quantifying the intensity of the product complex. The enzymes adsorbed on paper retained their functionality and selectivity. Adsorption on paper increased the enzyme thermal stability by 2-3 orders of magnitude compared to the same enzyme in solution. ALP and HRP enzymatic papers had half-lives of 533 h and 239 h at 23 degrees C, respectively. The thermal degradation of adsorbed enzyme was found to follow two sequential first-order reactions, indication of a reaction system. A complex pattern of enzyme was printed on paper using a thermal inkjet printer. Paper and inkjet printing are ideal material and process to manufacture low-cost-high volume bioactive surfaces. PMID:19775873

Khan, Mohidus Samad; Li, Xu; Shen, Wei; Garnier, Gil

2010-01-01

270

Fungal biodegradation and enzymatic modification of lignin  

PubMed Central

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

271

Glassy dynamics and enzymatic activity of lysozyme.  

PubMed

There has been much interest in the analogies between dynamic processes in proteins and in other complex systems such as viscous liquids and glasses. We have investigated the dynamics of protons along chains of hydrogen-bonded water molecules adsorbed on the surface of the globular protein lysozyme. The hydration dependence of the dielectric relaxation time is fitted by a modified Vogel-Fulcher-Tamman equation, in which the variable temperature has been replaced with hydration. We find that the relaxation time diverges at a singular hydration that coincides with the critical water content required to trigger lysozyme enzymatic activity. This surprising correlation suggests a direct coupling between protein function and glasslike behavior, with possible implications for the turnover number of the enzyme. PMID:11735996

Pizzitutti, F; Bruni, F

2001-11-01

272

Heavy atom isotope effects on enzymatic reactions  

NASA Astrophysics Data System (ADS)

The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

Paneth, Piotr

1994-05-01

273

Proteinuria: an enzymatic disease of the podocyte?  

PubMed Central

Proteinuria is a major health-care problem that affects several hundred million people worldwide. Proteinuria is a cardinal sign and a prognostic marker of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Microalbuminuria is the earliest cue of renal complications of diabetes, obesity, and the metabolic syndrome. It can often progress to overt proteinuria that in 10–50% of patients is associated with the development of chronic kidney disease, ultimately requiring dialysis or transplantation. Therefore, reduction or prevention of proteinuria is highly desirable. Here we review recent novel insights into the pathogenesis and treatment of proteinuria, with a special emphasis on the emerging concept that proteinuria can result from enzymatic cleavage of essential regulators of podocyte actin dynamics by cytosolic cathepsin L (CatL), resulting in a motile podocyte phenotype. Finally, we describe signaling pathways controlling the podocyte actin cytoskeleton and motility and how these pathways can be manipulated for therapeutic benefit.

Mundel, Peter; Reiser, Jochen

2014-01-01

274

Recent development of miniatured enzymatic biofuel cell  

NASA Astrophysics Data System (ADS)

Enzymatic biofuel cells (EBFCs) that oxidize biological fuels using enzyme-modified electrodes are considered a promising candidate for implantable power sources. However, there are still challenges to overcome before biofuel cells become competitive in any practical applications. Currently, the short lifespan of the catalytic enzymes and poor power density are the most critical issues in developing EBFCs. In this paper, we will review the recent development of biofuel cells and highlight the progress in Carbon-microelectromechanical system (C-MEMS) based micro biofuel cells by both computational modeling and experimental work. Also, our effort on utilizing a covalent immobilization technique for the attachment of enzymes onto the substrate which is expected to increase the enzyme loading efficiency and the power density of devices is discussed in this paper.

Song, Yin; Penmatsa, Varun; Wang, Chunlei

2011-05-01

275

Herpes simplex virus 1 protein kinase Us3 phosphorylates viral dUTPase and regulates its catalytic activity in infected cells.  

PubMed

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase. PMID:24173231

Kato, Akihisa; Tsuda, Shumpei; Liu, Zhuoming; Kozuka-Hata, Hiroko; Oyama, Masaaki; Kawaguchi, Yasushi

2014-01-01

276

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells  

PubMed Central

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.

Kato, Akihisa; Tsuda, Shumpei; Liu, Zhuoming; Kozuka-Hata, Hiroko; Oyama, Masaaki

2014-01-01

277

?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis  

PubMed Central

Background: The content of icaritin and genistein in herba is very low, preparation with relatively large quantities is an important issue for extensive pharmacological studies. Objective: This study focuses on preparing and enzymic hydrolysis of flavonoid glycosides /?-cyclodextrin inclusion complex to increase the hydrolysis rate. Materials and Methods: The physical property of newly prepared inclusion complex was tested by differential scanning calorimetry (DSC). The conditions of enzymatic hydrolysis were optimized for the bioconversion of flavonoid glycosides /?-cyclodextrin inclusion complex by mono-factor experimental design. The experiments are using the icariin and genistein as the model drugs. Results: The solubility of icariin and genistein were increased almost 17 times from 29.2 ?g/ml to 513.5 ?g/ml at 60°C and 28 times from 7.78 ?g/ml to 221.46 ?g/ml at 50°C, respectively, demonstrating that the inclusion complex could significantly increase the solubility of flavonoid glycosides. Under the optimal conditions, the reaction time of icariin and genistin decreased by 68% and 145%, when compared with that without ?-CD inclusion. By using this enzymatic condition, 473 mg icaritin (with the purity of 99.34%) and 567 mg genistein(with the purity of 99.46%), which was finally determined by melt point, ESI-MS, UV, IR, 1H NMR and 13C NMR, was obtained eventually by transforming the inclusion complex(contains 1.0 g substrates). Conclusion: This study can clearly indicate a new attempt to improve the speed of enzyme-hydrolysis of poorly water-soluble flavonoid glycosides and find a more superior condition which is used to prepare icaritin and genistein.

Jin, Xin; Zhang, Zhen-hai; Sun, E.; Jia, Xiao-Bin

2013-01-01

278

Post-Translational Phosphorylation of Serine 74 of Human Deoxycytidine Kinase Favors the Enzyme Adopting the Open Conformation Making It Competent for Nucleoside Binding and Release  

SciTech Connect

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon (UIC); (MXPL-G)

2012-03-26

279

Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release  

PubMed Central

Deoxycytidine kinase (dCK) uses either ATP or UTP as phosphoryl donors to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is situated distant to the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the enzyme adopting the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in close proximity to the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild type dCK and the S74E variant.

Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

2011-01-01

280

Regulation of VASP by phosphorylation: consequences for cell migration.  

PubMed

Phosphorylations control all aspects of vasodilator-stimulated phospho-protein (VASP) function. Mapped phosphorylation sites include Y39, S157, S239, T278, and S322, and multiple kinases have been shown to mediate their phosphorylation. Recently, Protein Kinase D1 (PKD1) as a direct kinase for S157 and S322 joined this group. While S157 phosphorylation generally seems to serve as a signal for membrane localization, phosphorylations at S322 or at S239 and T278 have opposite effects on F-actin accumulation. In migrating cells, S322 phosphorylation increases filopodia numbers and length, while S239/T278 phosphorylations decrease these and also disrupt formation of focal adhesions. Therefore, the kinases mediating these phosphorylations can be seen as switches needed to facilitate cell motility. PMID:24401601

Döppler, Heike; Storz, Peter

2013-01-01

281

Solution structure of calmodulin bound to the target peptide of endothelial nitric oxide synthase phosphorylated at Thr495.  

PubMed

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an ?-helix. The calcium affinity of the CaM-target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue. PMID:24495081

Piazza, Michael; Taiakina, Valentina; Guillemette, Simon R; Guillemette, J Guy; Dieckmann, Thorsten

2014-03-01

282

Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.  

PubMed Central

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.

Pillay, T S; Xiao, S; Olefsky, J M

1996-01-01

283

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria  

Microsoft Academic Search

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem

N. Irene Kavanagh; Edward K. Ainscow; Martin D. Brand

2000-01-01

284

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases  

Microsoft Academic Search

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat

Travis J. Jewett; Cheryl A. Dooley; David J. Mead; Ted Hackstadt

2008-01-01

285

Serine phosphorylation regulates paxillin turnover during cell migration  

Microsoft Academic Search

BACKGROUND: Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines

Nancy Abou Zeid; Ana-Maria Vallés; Brigitte Boyer

2006-01-01

286

Casein kinase 2 associates with and phosphorylates Dishevelled  

Microsoft Academic Search

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that

Karl Willert; Marcel Brink; Andreas Wodarz; Harold Varmus; Roel Nusse

1997-01-01

287

Protein kinase Akt/PKB phosphorylates heme oxygenase-1 in vitro and in vivo.  

PubMed

Heme oxygenase-1 (HO-1) is a stress response protein that protects cells against diverse noxious stimuli. Although regulation of HO-1 occurs mainly at the transcriptional level, its posttranslational modifications remain unexplored. We have identified a putative consensus sequence for phosphorylation by Akt/PKB of HO-1 at Ser188. Recombinant human and rat HO-1, but not mutant HO-1(S188A), are phosphorylated in vitro by Akt/PKB. Isotopic 32P-labeling of HEK293T cells confirmed that HO-1 is a phosphoprotein and that the basal HO-1 phosphorylation is increased by Akt1 activation. HO-1(S188D), a single point mutant equivalent to the phosphorylated protein, exhibited over 1.6-fold higher activity than wild type HO-1. Fluorescence resonance energy transfer (FRET) studies indicated that HO-1(S188D) bound to cytochrome P450 reductase (CPR) and biliverdin reductase (BVR) with a slightly lower Kd than wild-type HO-1. Although the changes in activity are small, this study provides the first evidence for a role of the survival kinase Akt in the regulation of HO-1. PMID:15581622

Salinas, Marta; Wang, Jinling; Rosa de Sagarra, María; Martín, Daniel; Rojo, Ana I; Martin-Perez, Jorge; Ortiz de Montellano, Paul R; Cuadrado, Antonio

2004-12-01

288

Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D-mediated Phosphorylation  

PubMed Central

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase III? and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

Nhek, Sokha; Ngo, Mike; Yang, Xuemei; Ng, Michelle M.; Field, Seth J.; Asara, John M.; Ridgway, Neale D.

2010-01-01

289

LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1).  

PubMed

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation. PMID:21283629

An, Hanbing; Lu, Xinyuan; Liu, Dan; Yarbrough, Wendell G

2011-01-01

290

Hits, Fhits and Nits: beyond enzymatic function.  

PubMed

We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions. PMID:21035495

Huebner, Kay; Saldivar, Joshua C; Sun, Jin; Shibata, Hidetaka; Druck, Teresa

2011-01-01

291

Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo  

SciTech Connect

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with {sup 32}P{sub i}. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The {sup 32}P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extend phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. The authors conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.

Ray, L.B.; Sturgill, T.W. (Univ. of Virginia School of Medicine, Charlottesville (USA))

1988-06-01

292

Novel reversible biotinylated probe for the selective enrichment of phosphorylated peptides from complex mixtures.  

PubMed

To improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotin-tag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fluorenyl methoxycarbonyl (4-carboxy Fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. Using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. Unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base liberates a covalently bound Gly-Cys analog of the peptide(s) of interest, exhibiting improved RP-HPLC retention and MS ionization properties compared with the precursor phosphopeptide sequence. The results obtained for a model peptide Akt-1 and two protein digests, demonstrated that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of fmol/microL. PMID:18359247

Jalili, Pegah R; Ball, Haydn L

2008-05-01

293

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.  

PubMed

Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant ?-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The ?-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased ?-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in ?-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased ?-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs. PMID:24033810

Madhavarao, Chikkathur N; Agarabi, Cyrus D; Wong, Lily; Müller-Loennies, Sven; Braulke, Thomas; Khan, Mansoor; Anderson, Howard; Johnson, Gibbes R

2014-01-01

294

Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence  

PubMed Central

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.

Plafker, Scott M.; Woods, Amina S.; Gibson, Wade

1999-01-01

295

Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells  

PubMed Central

The ability to make sensitive measurements of protein–protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 ?-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein–protein interactions in single cells with high signal discrimination.

Spotts, James M.; Dolmetsch, Ricardo E.; Greenberg, Michael E.

2002-01-01

296

Proteomic analysis of phosphorylation in cancer.  

PubMed

Constitutive activity of kinases is known to be crucial for a tumor to maintain its malignant phenotype, a phenomenon which is often referred to as oncogene addiction. The in-depth analysis of aberrant signaling pathways by the analysis of protein phosphorylation has become feasible through recent advances in proteomics technology. In this article we will review developments in the field of phosphoproteomics and its application in cancer research. The most widely used technologies for the generic enrichment of phosphopeptides are discussed as well as targeted approaches for the analysis of a specific subset of phosphopeptides. Validation experiments of phosphorylation sites using targeted mass spectrometry are also explained. Finally, we will highlight applications of phosphoproteomic technology in cancer research using cell lines and tissue. PMID:24666026

Ruprecht, Benjamin; Lemeer, Simone

2014-06-01

297

Uncoupling of mitochondrial oxidative phosphorylation by hexetidine.  

PubMed

To gain further insight into the biochemical properties of the antibacterial hexetidine, isolated rat liver mitochondria were added with this drug and investigation made of certain features related to mitochondrial bioenergetics. Hexetidine was found to cause oxidation of intramitochondrial pyridine nucleotides and stimulate the rate of oxygen uptake caused by respiratory substrates involving three, two and one site(s) of phosphorylation. Reversal of oxygen uptake inhibition by oligomycin was also determined. By investigating hexetidine effect on oxidative phosphorylation, hexetidine was found both to inhibit the rate of ATP synthesis and to cause ATP hydrolysis. Likewise, hexetidine capability to produce acidification of extramitochondrial medium and to collapse delta psi was also observed. The reported findings show that hexetidine exhibits uncoupling properties. PMID:3632700

D'Arcangelo, G; Barile, M; Passarella, S; Quagliariello, E

1987-09-15

298

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA.  

PubMed

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the (1)H,(15)N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate. PMID:23456038

Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

2013-04-01

299

Solid polymer electrolyte from phosphorylated chitosan  

NASA Astrophysics Data System (ADS)

Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component's composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it's characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10-6 S/cm up to 6.01 × 10-4 S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10-3 S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

Fauzi, Iqbal; Arcana, I. Made

2014-03-01

300

Enzymatic detection of troponin C and melittin bee  

NASA Astrophysics Data System (ADS)

One of the major goals of biosensor technology is to detect and quantify in detail analytes with very high accuracy. To achieve this, much of the emphasis in sensor fabrication has been laid on antibody-antigen interaction. The consequence of this focus of enzyme biosensor studies is the development of critical techniques which can be extended in the detection of Acute Myocardial Infarction (AMI). Biosensors for AMI have attracted considerable interest in the last few years since the monitoring of a specific substance is central in enzymatic reactions. This interest has led to the investigation of biochemical markers of myocardial injury. These biomarkers facilitate the diagnosis and treatment of patients with AMI. Serial measurements of biochemical markers are now universally accepted as an important determinant in AMI diagnosis. Due to their high sensitivity and specificity over other biomarkers, the troponins are the markers of choice for the diagnosis or exclusion of AMI. The present techniques used in the identification of the troponins are lengthy and require large amount of specimen solution. The present research is directed towards the identification of optical detection procedures that are compatible to the miniaturization. In the present study an effort has been made to study the antigen-antibody reaction of rabbit skeletal muscle troponin C (TnC) and bee venom melittin (ME). Fluorescence energy transfer experiments were done to investigate the Ca 2+ -dependant interaction of TnC-ME in a 1:1 complex. Experiments were also conducted on TnC-ME binding at different ratios. These results validate the biosensor technology and illustrate how a biosensor can be developed based on the study of interaction between monoclonal antibody and antigen reaction in real time. The reported experimental results provide valuable information that will be useful in the development of a biosensor for the detection of AMI.

Jeetender, Amritsar; Stiharu, Ion; Packirisamy, Muthukumaran

2005-04-01

301

Phosphorylation-Dependent Regulation of Ryanodine Receptors  

PubMed Central

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

302

Phosphorylation network rewiring by gene duplication  

PubMed Central

Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post-translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post-duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase–substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post-translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them.

Freschi, Luca; Courcelles, Mathieu; Thibault, Pierre; Michnick, Stephen W; Landry, Christian R

2011-01-01

303

Phosphorylation of proteins in Clostridium thermohydrosulfuricum.  

PubMed Central

Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by [gamma-32P]ATP of several endogenous proteins with Mrs between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of Mrs 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10 microM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 microM brain (but not spinach) calmodulin. Polyamines, including the "odd" polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32Pi. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro. Images

Londesborough, J

1986-01-01

304

Multistationarity in sequential distributed multisite phosphorylation networks.  

PubMed

Multisite phosphorylation networks are encountered in many intracellular processes like signal transduction, cell-cycle control, or nuclear signal integration. In this contribution, networks describing the phosphorylation and dephosphorylation of a protein at n sites in a sequential distributive mechanism are considered. Multistationarity (i.e., the existence of at least two positive steady state solutions of the associated polynomial dynamical system) has been analyzed and established in several contributions. It is, for example, known that there exist values for the rate constants where multistationarity occurs. However, nothing else is known about these rate constants. Here, we present a sign condition that is necessary and sufficient for multistationarity in n-site sequential, distributive phosphorylation. We express this sign condition in terms of linear systems, and show that solutions of these systems define rate constants where multistationarity is possible. We then present, for n?2, a collection of feasible linear systems, and hence give a new and independent proof that multistationarity is possible for n?2. Moreover, our results allow to explicitly obtain values for the rate constants where multistationarity is possible. Hence, we believe that, for the first time, a systematic exploration of the region in parameter space where multistationarity occurs has become possible. One consequence of our work is that, for any pair of steady states, the ratio of the steady state concentrations of kinase-substrate complexes equals that of phosphatase-substrate complexes. PMID:24048546

Holstein, Katharina; Flockerzi, Dietrich; Conradi, Carsten

2013-11-01

305

Hunting for Serine 276-Phosphorylated p65  

PubMed Central

The transcription factor nuclear factor kappaB (NF-?B) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-?B, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-?B subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.

Spooren, Anneleen; Kolmus, Krzysztof; Vermeulen, Linda; Van Wesemael, Karlien; Haegeman, Guy; Gerlo, Sarah

2010-01-01

306

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1  

PubMed Central

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.

Barisic, Sandra; Nagel, Anja C; Franz-Wachtel, Mirita; Macek, Boris; Preiss, Anette; Link, Gisela; Maier, Dieter; Hausser, Angelika

2011-01-01

307

Enzymatic production of hydrogen from glucose  

SciTech Connect

The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP{sup +} requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H{sub 2}ase) which catalyzes the evolution of H{sub 2}. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H{sub 2}ase yielding 17% of theoretical maximum expected. The cofactor NADP{sup +} for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally stable enzymes are underway.

Woodward, J. [Oak Ridge National Lab., TN (United States); Mattingly, S.M.

1995-06-01

308

Natural Product Sugar Biosynthesis and Enzymatic Glycodiversification**  

PubMed Central

Many biologically active small molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products, and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and/or alter the glycosylation patterns of natural products via metabolic pathway engineering and/or enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.

Thibodeaux, Christopher J.; Melancon, Charles E.; Liu, Hung-wen

2009-01-01

309

Ecological and enzymatic responses to petroleum contamination.  

PubMed

The changes in microbial ecology interpreted from taxonomic and functional genes and biological functions represented by urease and dehydrogenase activities were monitored in soil contaminated with different petroleum hydrocarbons including crude oil, diesel, n-hexadecane and poly-aromatic hydrocarbons (PAHs). It was shown that the presence of n-hexadecane stimulated the activity of indigenous microorganisms, especially alkane degrading bacteria, and led to over 20% degradation of n-hexadecane within one month. No obvious degradation of the other three types of petroleum hydrocarbons was observed. The stimulation effect was most marked in the soil spiked with a medium concentration (2500 mg kg(-1) dry soil) of n-hexadecane. However, the presence of PAHs completely inhibited the previously-mentioned bioactivities of the soil. The content of PAH degrading bacteria, however, increased more than 10-fold, indicating the selection effect of PAHs on soil bacteria. The impacts of diesel and crude oil on the microbial ecology and biological functions varied significantly with their concentration. The disclosure of the ecological and enzymatic responses could be helpful in soil bioremediation. PMID:24765642

Wu, Binbin; Lan, Tian; Lu, Diannan; Liu, Zheng

2014-05-28

310

Nanostructured Organometallic Polymers for Enzymatic Bioenergy  

NASA Astrophysics Data System (ADS)

The development of efficient enzymatic biofuel cell is a subject of considerable studies in past decades for potential applications such as biomedical devices and microchip systems. One of the key challenges in advancing the technology lies in the power densities of the system. Limitations have been arisen from the buried redox active sites within enzyme structure and poor interplay between redox reactions. In present study, a glucose oxidase is employed as a model enzyme and ferrocene-containing organometallic block copolymers are chosen for the electron mediators. Wiring of glucose oxidase into electrode surface was successfully achieved by cross-linked networks of organometallic polymers and remarkably, catalytic current densities of the fabricated electrodes have proven be a sensitive function of the morphologies of electron mediators. Different nanoscale morphologies, i.e., bicontinous structure, nanowires, and nanoparticles, have been derived and the use of bicontinous morphology confirms 2-50 times improved catalytic current response than the values obtained from other morphologies. The bio-sensing ability of the fabricated electrode with structural optimization was also exploited and good sensitivity is obtained at the physiological concentration of glucose in blood.

Park, Moon Jeong; Lee, Jungphil

2012-02-01

311

Enzymatic conversion of sucrose to hydrogen  

SciTech Connect

The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP{sup +}/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of mol of H{sub 2}/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP{sup +}. Although gluconic and inhibited both NADPH production and oxidation of GDH and hydrogenase, respectively, it was not the major cause of NADP{sup +} instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1.34 mol of H{sub 2}/mol of sucrose was obtained if GI was present in the reaction mixture.

Woodward, J.; Orr, M. [Oak Ridge National Lab., TN (United States). Chemical Technology Div.] [Oak Ridge National Lab., TN (United States). Chemical Technology Div.

1998-11-01

312

Enzymatic conversion of sucrose to hydrogen  

PubMed

The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP+/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of 1 mol of H2/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP+. Although gluconic acid inhibited both NADPH production and oxidation by GDH and hydrogenase, respectively, it was not the major cause of NADP+ instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1. 34 mol of H2/mol of sucrose was obtained if GI was present in the reaction mixture. PMID:9841653

Woodward; Orr

1998-11-01

313

The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue.  

PubMed

Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis. PMID:19074144

Veyron-Churlet, Romain; Molle, Virginie; Taylor, Rebecca C; Brown, Alistair K; Besra, Gurdyal S; Zanella-Cléon, Isabelle; Fütterer, Klaus; Kremer, Laurent

2009-03-01

314

NAD(+)-NADH Modelling Studies: An Enzymatic and Chemical Approach.  

National Technical Information Service (NTIS)

Chemical and enzymatic studies on the coenzyme NAD+ and its reduced form NADH are reported. In combination with specific enzymes (dehydrogenases) the coenzyme is involved in the reversible stereospecific dehydrogenation of many substrates. Major focus is ...

N. A. Beijer

1990-01-01

315

Enzymatic Activities of Cell-Free Extracts of 'Rickettsia typhi'.  

National Technical Information Service (NTIS)

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), mode...

J. C. Coolbaugh J. J. Progar E. Weiss

1976-01-01

316

Enzymatic Methods of Making Fructose and Selected Other Work.  

National Technical Information Service (NTIS)

Progress is reported in programs directed toward the enzymatic isomerization of glucose to fructose and the design of a NAD-dehydrogenase reactor. The fructose section reports work on basic equilibrium and kinetic data, characteristics of whole cells entr...

A. Emery V. W. Rodwell H. C. Lim F. E. Regnier D. R. Schneider

1974-01-01

317

MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)  

EPA Science Inventory

An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

318

Quantitative analysis of the effect of salt concentration on enzymatic catalysis.  

PubMed

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt-rate profiles: k(cat)/K(M) = (k(cat)/K(M))(MAX)/[1+([Na+]/K[Na+])(n')], where (k(cat)/K(M))(MAX) is the physical limit of k(cat)/K(M), K(Na+) is the salt concentration at which k(cat)/K(M) = (k(cat)/K(M))(MAX)/2, and -n' is the slope of the linear region in a plot of log(k(cat)/K(M)) versus log [Na+]. The value of n' is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k(cat)/K(M) values that can exceed 10(9) M(-1) s(-1). Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n'. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n'. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n', as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt-rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis. PMID:11707126

Park, C; Raines, R T

2001-11-21

319

Enzymatic synthesis and chemical recycling of poly(carbonate-urethane).  

PubMed

Novel enzymatically recyclable poly(carbonate-urethane) consisting of a diurethane moiety as a hard segment and a carbonate linkage as an enzymatically cleavable unit was prepared by the polycondensation of biodegradable diurethanediol and diethyl carbonate using lipase. The produced poly(carbonate-urethane) was readily transformed by lipase into the corresponding cyclic oligomers which were more easily repolymerized by lipase to produce a higher molecular weight poly(carbonate-urethane) than that of the parent poly(carbonate-urethane). PMID:15468266

Soeda, Yasuyuki; Toshima, Kazunobu; Matsumura, Shuichi

2004-08-01

320

Enzymatic Treatment of Wool Fabric : Effects of the Surfactants  

Microsoft Academic Search

By use of the commercial protease, the wool fabric has been treated enzymatically. The modified form of the Michaelis-Menten equation has been used and the maximal velocity and the half-saturation constant, V . and k'm, were calculated. The effects of different types of surfactants (cationic 'CAS', non-ionic 'NNS', anionic 'ANS', amphoteric 'AMS') on the rate of enzymatic reaction were studied.

Nahid Hemmati Nejad; Shahla Salahshour Kordestani; Farzaneh Vahabzadeh

321

Changes in lipid composition of Photosystem 1 particles from thylakoids phosphorylated under reductive or anaerobic conditions.  

PubMed

Changes in lipid composition of Photosystem 1 (PS 1) particles isolated from thylakoids phosphorylated under reductive or anaerobic conditions have been studied. Under reductive conditions, there was an increase in monogalactosyldiacylglycerol containing highly saturated fatty acids and phosphatidylglycerol containing transhexadecenoic fatty acid. Under anaerobic conditions, the amount of all lipid classes was increased. As we have shown earlier (S. V. Manuilskaya, O. I. Volovik, A. I. Mikhno, A. I. Polischuk and S. M. Kochubey (1990) Photosynthetica 24: 419-423) these changes were due to a co-migration of some lipid species and light-harvesting chlorophyll a/b complex LHC II from PS 2 to PS 1. These data allow us to conclude that LHC II consists of the lipoproteins containing specific lipids. Different composition of lipids co-migrating with LHC II under various conditions of phosphorylation might be caused by the variety of LHC II subpopulations transferred under each reductive condition. PMID:24306845

Manuilskaya, S V; Volovik, O I; Kochubey, S M

1995-03-01

322

Role of substrate unbinding in Michaelis-Menten enzymatic reactions.  

PubMed

The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover--turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle. PMID:24616494

Reuveni, Shlomi; Urbakh, Michael; Klafter, Joseph

2014-03-25

323

Enzymatically induced motion at nano- and micro-scales.  

PubMed

In contrast to adenosine triphosphate (ATP)-dependent motor enzymes, other enzymes are little-known as "motors" or "pumps", that is, for their ability to induce motion. The enhanced diffusive movement of enzyme molecules, the self-propulsion of enzyme-based nanomotors, and liquid pumping with enzymatic micropumps were indeed only recently reported. Enzymatically induced motion can be achieved in mild conditions and without the use of external fields. It is thus better suited for use in living systems (from single-cell to whole-body) than most other ways to achieve motion at small scales. Enzymatically induced motion is thus not only new but also important. Therefore, the present work reviews the most significant discoveries in enzymatically induced motion. As we will learn, freely diffusing enzymes enhance their diffusive movement by nonreciprocal conformational changes which parallel their catalytic cycles. Meanwhile, enzyme-modified nano- and micro-objects turn chemical energy into kinetic energy through mechanisms such as bubble recoil propulsion, self-electrophoresis, and self-diffusiophoresis. Enzymatically induced motion of small objects ranges from enhanced diffusive movement to directed motion at speeds as high as 1 cm s(-1). In spite of the progress made in understanding how the energy of enzyme reactions is turned into motion, most enzymatically powered devices remain inefficient and need improvements before we will witness their application in real world environments. PMID:24931666

Gáspár, Szilveszter

2014-06-26

324

Tyrosine phosphorylation of CD13 regulates inflammatory cell-cell adhesion and monocyte trafficking.  

PubMed

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein ?-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions. PMID:23997214

Subramani, Jaganathan; Ghosh, Mallika; Rahman, M Mamunur; Caromile, Leslie A; Gerber, Claire; Rezaul, Karim; Han, David K; Shapiro, Linda H

2013-10-01

325

Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation.  

PubMed

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase. PMID:17932035

Lecchi, Silvia; Nelson, Clark J; Allen, Kenneth E; Swaney, Danielle L; Thompson, Katie L; Coon, Joshua J; Sussman, Michael R; Slayman, Carolyn W

2007-12-01

326

Effects of cadmium on enzymatic and non-enzymatic antioxidative defences of rice (Oryza sativa L.).  

PubMed

The effects of 60-d cadmium (Cd) exposure on enzymatic and non-enzymatic antioxidative system of Oryza sativa L. seedlings at tillering stage were studied using soil culture experiment. Research findings showed that chlorophyll content of Oryza sativa L. declined with the increase in soil metal concentration. Cd pollution induced the antioxidant stress by inducing O2(-1) and H2O2, which increased in plants; at the same time, MDA as the final product of peroxidation of membrane lipids, accumulated in plant. The antioxidant enzyme system was initiated under the Cd exposure, i.e. almost all the activities of superoxide dismutase (SOD), peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase were elevated both in leaves and roots. The non-protein thiols including phytochelatins and glutathione to scavenge toxic free radicals caused by Cd stress was also studied. The contents of phytochelatins and glutathione were about 3.12-6.65-fold and 3.27-10.73-fold in leaves, against control; and the corresponding values were about 3.53-9.37-fold and 1.41-5.11-fold in roots, accordingly. PMID:23819293

Yu, Fangming; Liu, Kehui; Li, Mingshun; Zhou, Zhenming; Deng, Hua; Chen, Bin

2013-01-01

327

Enzymatic membrane reactors for biodegradation of recalcitrant compounds. Application to dye decolourisation  

Microsoft Academic Search

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages

C López; I Mielgo; M. T Moreira; G Feijoo; J. M Lema

2002-01-01

328

Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex  

NASA Technical Reports Server (NTRS)

The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

Kowalczyk, S.; Bandurski, R. S.

1990-01-01

329

Enzymatic reduction of 3-nitrotyrosine generates superoxide.  

PubMed

Spin-trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) was used to demonstrate that 3-nitrotyrosine (nitrotyrosine) promotes the formation of substantial amounts of reactive oxygen species (O2.- and *OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Spin adduct formation is strongly inhibited by the presence of superoxide dismutase (SOD); spin adduct formation requires aerobic conditions. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide, results in a similar generation of O2*- and *OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferricytochrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis-Menten kinetics and Km's of 104 +/- 14 and 0.78 +/- 0.11 microM, respectively. No stimulation of acetylated ferricytochrome c reduction is observed in the presence of SOD. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferricytochrome c reduction by nitrotyrosine. Nitration of two tyrosines (of four) within the sequence of the 6.5-kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2*- generation implying steric restrictions for BPTI-reductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical (ArNO2*-) which is then oxidized by molecular oxygen to yield O2*- and regenerate ArNO2. Thus, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling. PMID:9585480

Krainev, A G; Williams, T D; Bigelow, D J

1998-05-01

330

Enzymatic sequencing of partially acetylated chitosan oligomers.  

PubMed

Chitosan oligosaccharides have diverse biological activities with potentially valuable applications, for example, in the fields of medicine and agriculture. These functionalities are thought to depend on their degree of polymerization and acetylation, and possibly on specific patterns of acetylation. Chitosan oligomers with fully defined architecture are difficult to produce, and their complete analysis is demanding. Analysis is typically done using MS or NMR, requiring access to expensive infrastructure, and yielding unequivocal results only in the case of rather small oligomers. We here describe a simple and cost-efficient method for the sequencing of ?g amounts of chitosan oligosaccharides which is based on the sequential action of two recombinant glycosidases, namely an exo-?-N-acetylhexosaminidase (GlcNAcase) from Bacillus subtilis 168 and an exo-?-d-glucosaminidase (GlcNase) from Thermococcus kodakarensis KOD1. Starting from the non-reducing end, GlcNAcase and GlcNase specifically remove N-acetyl glucosamine (A) and glucosamine (D) units, respectively. By the sequential addition and removal of these enzymes in an alternating way followed by analysis of the products using high-performance thin-layer chromatography, the sequence of chitosan oligosaccharides can be revealed. Importantly, both enzymes work under identical conditions so that no buffer exchange is required between steps, and the enzyme can be removed conveniently using simple ultra-filtration devices. As proof-of-principle, the method was used to sequence the product of enzymatic deacetylation of chitin pentamer using a recombinant chitin deacetylase from Vibrio cholerae which specifically removes the acetyl group from the second unit next to the non-reducing end of the substrate, yielding mono-deacetylated pentamer with the sequence ADAAA. PMID:24824785

Hamer, Stefanie Nicole; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

2014-06-17

331

Enzymatic production of ceramide from sphingomyelin.  

PubMed

Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potentials in cosmetic and pharmaceutical industries such as in hair and skin care products. Chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient production methods are of great interest. Present study was the first attempt to perform a systematic study on the production of ceramide through enzymatic hydrolysis of sphingomyelin. Sphingomyelin hydrolysis proved to be more efficient in two-phase (water:organic solvent) system than in one-phase (water-saturated organic solvent) system. Among the screened phospholipase C, the Clostridium perfringens enzyme had the highest sphingomyelin conversion rate, with very small temperature dependence. Addition of ethanol to the system markedly enhanced the rate of ceramide formation, and a mixture of ethylacetate:hexane (50:50) was the best organic solvent tested. Other factors such as (NH(4))(2)SO(4), NaCl and CaCl(2) were also tested but excluded for further consideration. On the basis of the initial experiments, the reaction system was optimized using response surface methodology including five factors (enzyme amount, water amount, ethanol amount, reaction time and the hexane ratio of organic solvent). Water content and enzyme amount was shown to have the most significant influence on the hydrolysis reaction in the fitted quadratic model. The efficiency of sphingomyelin hydrolysis was dramatically improved through system evaluation and optimization, with the optimal conditions at 75 min reaction time, 3 Uml(-1) enzyme amount, 6% water amount, 1.8% ethanol amount and 46% hexane in ethylacetate. PMID:16337303

Zhang, Long; Hellgren, Lars I; Xu, Xuebing

2006-05-01

332

Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles  

SciTech Connect

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

Franke, Claudia [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Matschl, Urte [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Bruns, Michael [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany)]. E-mail: mbruns@hpi.uni-hamburg.de

2007-03-01

333

Regulation of renal fibrosis by smad3 thr388 phosphorylation.  

PubMed

Transforming growth factor-? (TGF-?) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-?1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-?-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-?/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease. PMID:24485922

Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

2014-04-01

334

Roles of subunit phosphorylation in regulating glutamate receptor function.  

PubMed

Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-d-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluRs The common kinases include protein kinase A, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer's disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

Wang, John Q; Guo, Ming-Lei; Jin, Dao-Zhong; Xue, Bing; Fibuch, Eugene E; Mao, Li-Min

2014-04-01

335

Mitotic phosphorylation of histone H3 threonine 80.  

PubMed

The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells. PMID:24275038

Hammond, Sharra L; Byrum, Stephanie D; Namjoshi, Sarita; Graves, Hillary K; Dennehey, Briana K; Tackett, Alan J; Tyler, Jessica K

2014-02-01

336

Respiration and Oxidative Phosphorylation in Treponema pallidum  

PubMed Central

Exogenous and endogenously generated reduced pyridine nucleotides caused marked stimulation of O2 uptake when added to treponemal cell-free extracts, which indicated that terminal electron transport was coupled to the consumption of O2. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) was shown to correlate stoichiometrically with O2 reduction, suggesting that NADH was being oxidized through a mainstream respiratory chain dehydrogenase. Oxygen evolution in treponemal extracts was observed after the completion of O2 uptake which was stimulated by exogenous NADH and endogenously generated reduced NAD phosphate. Oxygen evolution was inhibited by both cyanide and pyruvate, which was consistent with O2 release from H2O2 by catalase. The addition of exogenous H2O2 to treponemal extracts caused rapid O2 evolution characteristic of a catalase reaction. A spectrophotometric assay was used to measure ATP formation in T. pallidum cell-free extracts that were stimulated with NADH. P/O ratios from 0.5 to 1.1 were calculated from the amounts of ATP formed versus NADH oxidized. Phosphorylating activity was dependent on Pi concentration and was sensitive to cyanide, N, N?-dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenyl hydrazone. Adenine nucleotide pools of T. pallidum were measured by the firefly luciferin-luciferase assay. Shifts in adenine nucleotide levels upon the addition of NADH to cell-free extracts were impossible to evaluate due to the presence of NAD+ nucleosidase. However, when whole cells, previously incubated under an atmosphere of 95% N2-5% CO2, were sparged with air, ATP and ADP levels increased, while AMP levels decreased. The shift was attributed to both oxidative phosphorylation and to the presence of an adenylate kinase activity. T. pallidum was also found to possess an Mg2+ - and Ca2+ -stimulated ATPase activity which was sensitive to N, N? -dicyclohexylcarbodiimide. These data indicated a capability for oxidative phosphorylation by T. pallidum.

Lysko, Paul G.; Cox, C. D.

1978-01-01

337

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.  

PubMed

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function. PMID:8382990

Bork, P; Sander, C; Valencia, A

1993-01-01

338

Silicon nanowires as a rechargeable template for hydride transfer in redox biocatalysis  

NASA Astrophysics Data System (ADS)

We report a new possible application of hydrogen-terminated silicon nanowires (H-SiNWs) as a rechargeable template for hydride transfer in redox biocatalysis. H-SiNWs transfer hydride efficiently to regenerate NADH by oxidizing Si-Hx bonds. The oxidized H-SiNWs were readily recharged for the continuous regeneration of NADH and enzymatic reactions.

Lee, Hwa Young; Kim, Jae Hong; Son, Eun Jin; Park, Chan Beum

2012-11-01

339

Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.  

PubMed

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

2000-04-01

340

Can You Hear Me Now? Regulating Transcriptional Activators by Phosphorylation  

NSDL National Science Digital Library

Extracellular signals often modulate the expression of specific genetic programs by triggering the phosphorylation of relevant transcription factors (TFs). Phosphorylation in turn regulates such TFs by altering their cellular localization, DNA binding affinity, or transcriptional activity. Structural approaches have revealed how phosphorylation turns some TFs on or off; but less is known about how phosphorylation regulates other transcription factors in a graded manner that depends on signal intensity. A recent paper by Graves and colleagues reveals how a group of phosphorylation sites in Ets-1 regulates its DNA binding activity. Their studies provide new insight into the importance of multisite phosphorylation for the graded regulation of transcription and highlight the involvement of allosteric mechanisms in this process.

Kevin H. Gardner (University of Texas Southwestern Medical Center;Departments of Biochemistry and Pharmacology REV); Marc Montminy (The Salk Institute for Biological Studies; REV)

2005-09-13

341

A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes.  

PubMed

Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 ?W cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 k? load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22200380

Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal

2012-10-01

342

Modulation of enzymatic activity of Src-family kinases in bovine T cells transformed by Theileria parva.  

PubMed

After infection with sporozoites of the protozoon Theileria parva (Tp) bovine T cells are readily transformed to permanent growth in vivo and in vitro. Their transformed state depends on the constant presence of the parasite but membrane signals remain important. Non-receptor tyrosine kinases play a critical role in the transduction of membrane signals in haematopoietic cells. We have investigated Src-family kinases in bovine T cells transformed by Tp. The T cell receptor-associated tyrosine kinase p60fyn had high activity in all cell lines tested. In addition, weak phosphorylation of 2 novel bands was observed associated with Fyn. In contrast to Fyn, enzymatic activity of p56lck, which in T cells has an essential role in signalling, was low. Furthermore, 1 of 3 Tp transformed cell lines was completely devoid of p56lck indicating that the enzyme is not necessary for the Tp dependent growth of the T cells. In addition to p60fyn and p56lck weak enzymatic activity of 1 splice variant of p53/56lyn was observed after infection of T cells with Tp. These data show that growth transformation by Tp influences kinase activity in bovine T cells. However, they also prove that p56lck does not play an essential role in the transformation mechanism. PMID:9778632

Fich, C; Klauenberg, U; Fleischer, B; Bröker, B M

1998-08-01

343

Heat Transfer  

NSDL National Science Digital Library

Students explore heat transfer and energy efficiency using the context of energy efficient houses. They gain a solid understanding of the three types of heat transfer: radiation, convection and conduction, which are explained in detail and related to the real world. They learn about the many ways solar energy is used as a renewable energy source to reduce the emission of greenhouse gasses and operating costs. Students also explore ways in which a device can capitalize on the methods of heat transfer to produce a beneficial result. They are given the tools to calculate the heat transferred between a system and its surroundings.

Integrated Teaching And Learning Program

344

Phosphorylated silica nanotubes: preparation and characterization  

NASA Astrophysics Data System (ADS)

Recently, the strategy of doping inorganic particles into polymer membranes to modify them has been studied intensively. However, these inorganic particles have a disadvantage without being in good compatibility with the polymers. To enhance the compatibility between inorganic particles and polymers, phosphorylated silica nanotubes (PSNTs) with specific high ratios of length to diameter are prepared. Silica nanotubes (SNTs) are prepared through the hydrolysis of tetraethyl orthosilicate in a mixture of aqueous ammonia and dl-tartaric acid, then PSNTs are obtained by silylation and phosphorylation modifications. The optimum synthesis conditions of PSNTs are explored; in addition, the as-prepared PSNTs are characterized by Fourier transform infrared, transmission electron microscope, BET, x-ray photoelectron spectroscopy analysis and thermogravimetric analysis. The results indicate that the ratio of length to diameter of the PSNTs is approximately 20, the thickness of the tube wall is 20 nm, the specific surface area of the PSNTs is 460.2 m2 g-1, the inner diameter of the PSNTs is 76 nm, many mesopores are distributed in the tube walls of the PSNTs, and the PSNTs have numerous hydroxyl active sites along their length direction. Therefore, PSNTs are desirable as suitable fillers of polymer membranes.

Zhang, Yuqing; Xu, Yan; Lu, Yiren; Zhao, Lili; Song, Lixin

2013-08-01

345

Phosphorylation regulates coilin activity and RNA association  

PubMed Central

Summary The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.

Broome, Hanna J.; Carrero, Zunamys I.; Douglas, Heather E.; Hebert, Michael D.

2013-01-01

346

Phosphorylation regulates coilin activity and RNA association.  

PubMed

The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation. PMID:23616925

Broome, Hanna J; Carrero, Zunamys I; Douglas, Heather E; Hebert, Michael D

2013-04-15

347

Phosphorylated TDP-43 in frontotemporal lobar degeneration and ALS  

PubMed Central

Objective TDP-43 is deposited as cytoplasmic and intranuclear inclusions in brains of subjects with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43. Methods We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy and immunoblots. Additionally, we performed investigations aimed at identifying the responsible kinases and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. Results We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially-available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15 nm diameter, and on immunoblots recognized hyperphosphorylated TDP-43 at 45 kDa, with additional 22–28 kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase 1 and 2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. Interpretation These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders.

Hasegawa, Masato; Arai, Tetsuaki; Nonaka, Takashi; Kametani, Fuyuki; Yoshida, Mari; Hashizume, Yoshio; Beach, Thomas G.; Buratti, Emanuele; Baralle, Francisco; Morita, Mitsuya; Nakano, Imaharu; Oda, Tatsuro; Tsuchiya, Kuniaki; Akiyama, Haruhiko

2009-01-01

348

Computational enzymatic catalysis--clarifying enzymatic mechanisms with the help of computers.  

PubMed

Enzymes play a biologically essential role in performing and controlling an important share of the chemical processes occurring in life. However, despite their critical role in nature, attaining a clear understanding of the way an enzyme acts, i.e. its catalytic mechanism, is a cumbersome task that requires the cooperative efforts of a large number of different scientific techniques. Computational methods offer a particularly insightful way to study such mechanisms, always beautifully complementing the information arising from experimental techniques and working as an excellent alternative for assessing the viability of different mechanistic proposals. This review highlights two important computational strategies to study enzymatic catalysis - the cluster modeling approach and the hybrid quantum mechanical/molecular mechanical (QM/MM) method - complemented with a selection of hand-picked examples of our own work. PMID:22870506

Sousa, Sérgio Filipe; Fernandes, Pedro Alexandrino; Ramos, Maria João

2012-09-28

349

Enzymatic and non-enzymatic activities of SHIP-1 in signal transduction and cancer.  

PubMed

PI3K cascade is a central signaling pathway regulating cell proliferation, growth, differentiation, and survival. Tight regulation of the PI3K signaling pathway is necessary to avoid aberrant cell proliferation and cancer development. Together with SHIP-1, the inositol phosphatases PTEN and SHIP-2 are the gatekeepers of this pathway. In this review, we will focus on SHIP-1 functions. Negative regulation of immune cell activation by SHIP-1 is well characterized. Besides its catalytic activity, SHIP-1 also displays non-enzymatic activity playing role in several immune pathways. Indeed, SHIP-1 exhibits several domains that mediate protein-protein interaction. This review emphasizes the negative regulation of immune cell activation by SHIP-1 that is mediated by its protein-protein interaction. PMID:21672530

Condé, Claude; Gloire, Geoffrey; Piette, Jacques

2011-11-15

350

Enzymatic accessibility of fiber hemp is enhanced by enzymatic or chemical removal of pectin.  

PubMed

Pectinolytic enzymes, steam explosion and alkaline treatment were used to assess the role of pectin for the accessibility of hydrolytic enzymes in the enzymatic hydrolysis of biomass. Hemp (Cannabis sativa L.), a potential energy crop especially in boreal climate with a low need of fertilizers, was used in the study either as untreated or anaerobically preserved raw material. Addition of pectinases increased the hydrolysis yield by 26%, 54%, and 64% from the theoretical carbohydrates of untreated, acid, and alkali-preserved materials, respectively. Steam explosion and hot alkali treatment increased the conversion of the total carbohydrates by 78% and 60%, respectively, compared to the untreated hemp. Elevated separation of cells within the hemp stalk tissues and an increased surface area was revealed after hot alkali or pectinase treatments, contributing to the increased conversion to sugars by commercial enzymes. PMID:22237172

Pakarinen, A; Zhang, J; Brock, T; Maijala, P; Viikari, L

2012-03-01

351

The Importance of the Interaction of CheD with CheC and the Chemoreceptors Compared to Its Enzymatic Activity during Chemotaxis in Bacillus subtilis  

PubMed Central

Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase. CheD also binds CheC, and the strength of this interaction is increased by phosphorylated CheY. CheC is believed to control the binding of CheD to the receptors in response to the levels of phosphorylated CheY. In addition to their role in adaptation, CheC and CheD also have separate enzymatic functions. CheC is a CheY phosphatase and CheD is a receptor deamidase. Previously, we demonstrated that CheC’s phosphatase activity plays a minor role in chemotaxis whereas its ability to bind CheD plays a major one. In the present study, we demonstrate that CheD’s deamidase activity also plays a minor role in chemotaxis whereas its ability to bind CheC plays a major one. In addition, we quantified the interaction between CheC and CheD using surface plasmon resonance. These results suggest that the most important features of CheC and CheD are not their enzymatic activities but rather their roles in adaptation.

Allen, George M.; Walukiewicz, Hanna E.; Rao, Christopher V.; Ordal, George W.

2012-01-01

352

Effect of caspase cleavage-site phosphorylation on proteolysis.  

PubMed Central

Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1' positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation.

Tozser, Jozsef; Bagossi, Peter; Zahuczky, Gabor; Specht, Suzanne I; Majerova, Eva; Copeland, Terry D

2003-01-01

353

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding*  

PubMed Central

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (?)-2?-carbomethoxy-3?-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.

Moritz, Amy E.; Foster, James D.; Gorentla, Balachandra K.; Mazei-Robison, Michelle S.; Yang, Jae-Won; Sitte, Harald H.; Blakely, Randy D.; Vaughan, Roxanne A.

2013-01-01

354

Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters  

SciTech Connect

The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with ..gamma..-/sup 32/P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity.

Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

1986-03-05

355

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.  

PubMed Central

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.

Amankwa, L. N.; Harder, K.; Jirik, F.; Aebersold, R.

1995-01-01

356

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.  

PubMed

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates. PMID:6316265

Billich, A; Stockhove, U; Witzel, H

1983-11-11

357

Sirt3 regulates metabolic flexibility of skeletal muscle through reversible enzymatic deacetylation.  

PubMed

Sirt3 is an NAD(+)-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1? subunit of PDH (PDH E1?). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1? subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation. PMID:23835326

Jing, Enxuan; O'Neill, Brian T; Rardin, Matthew J; Kleinridders, André; Ilkeyeva, Olga R; Ussar, Siegfried; Bain, James R; Lee, Kevin Y; Verdin, Eric M; Newgard, Christopher B; Gibson, Bradford W; Kahn, C Ronald

2013-10-01

358

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.  

PubMed Central

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.

Billich, A; Stockhove, U; Witzel, H

1983-01-01

359

Non-enzymatic glycation of antithrombin III in vitro.  

PubMed

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function. PMID:3349100

Sakurai, T; Boissel, J P; Bunn, H F

1988-03-17

360

Absolute measurement of phosphorylation levels in a biological membrane using atomic force microscopy: the creation of phosphorylation maps.  

PubMed

We show that it is possible to produce phosphorylation difference maps of biological membranes under conditions which reflect those in vivo and in which proteins remain functional. We also demonstrate that absolute levels of phosphorylation are retrieved through the application of an appropriate calibration method. Finally we show that the kinetics of phosphorylation/dephosphorylation can also be monitored. These methods are demonstrated on photosynthetic membranes from higher plants, for which protein phosphorylation is the dominant regulatory mechanism. We show directly that the most recent estimates of the phosphorylation levels in this system are reasonably accurate. Phosphorylation difference maps show that the distribution of phosphates is not even, with significantly higher levels at the membrane margins and patches of high phosphate density next to patches of low charge density. PMID:12093269

Liou, Je-Wen; Mulet, Xavier; Klug, David R

2002-07-01

361

Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domain  

PubMed Central

Background Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. Results Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly. Conclusion Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

2012-01-01

362

A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway.  

PubMed

Plants are able to integrate exogenous 1-deoxy-D-xylulose (DX) into the 2C-methyl-D-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-D-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing D-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as "xylulose kinase" might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J

2006-10-01

363

A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1  

PubMed Central

Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.

Hemmerlin, Andrea; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-Francois; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

2006-01-01

364

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells  

PubMed Central

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2?min in cells seeded on low fibronectin concentration ([FN]) and 4.3?min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v?3, but not in those by integrin ?5?1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

2014-01-01

365

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells.  

PubMed

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2?min in cells seeded on low fibronectin concentration ([FN]) and 4.3?min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v?3, but not in those by integrin ?5?1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics. PMID:25056908

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y-S; Chien, Shu; Wang, Yingxiao

2014-01-01

366

Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.  

PubMed Central

Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components.

Detwiler, P B; Ramanathan, S; Sengupta, A; Shraiman, B I

2000-01-01

367

An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.  

PubMed

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H(2)(18)O or (18)O(2). There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass. PMID:20929773

Vaaje-Kolstad, Gustav; Westereng, Bjørge; Horn, Svein J; Liu, Zhanliang; Zhai, Hong; Sørlie, Morten; Eijsink, Vincent G H

2010-10-01

368

PKC?-dependent phosphorylation of the glycine transporter 1  

PubMed Central

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [32P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40% -inhibition on Vmax was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKC?/? inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKC? inhibitors (PKC? inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that phosphorylation that conventional PKC?/? regulates the uptake of glycine, whereas PKC? is responsible for GlyT1 phosphorylation.

Vargas-Medrano, Javier; Castrejon-Tellez, Vicente; Fernando, Plenge; Ramirez, Ivan; Miranda, Manuel

2011-01-01

369

Enzymatic catalysis via liquid-liquid interfaces.  

PubMed

Enzymes acting in a biphasic liquid of water and organic solvent may show catalytic activity dependent on the amount of phase interface available. Such an effect may be caused by several mechanisms. For example, for hydroxynitrile lyase from Prunus amygdalus, substrate mass transfer limitation has been advocated, but also adsorption of the enzyme on the interface. In this commentary it is shown that often these two mechanisms will have qualitatively similar consequences. The reaction rate will be influenced by the organic substrate concentration, by the initial enzyme concentration, and by the volume-specific interfacial area, and these influences will be linear at low values but reach a saturation level at high values. To rule out any of the models, their quantitative mathematical descriptions should be compared, taking into account that both models may be valid simultaneously. PMID:12800131

Straathof, Adrie J J

2003-08-20

370

Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: Design and characterization of the enzymatic properties.  

PubMed

Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded. PMID:24594066

Shadrina, Olga; Krotova, Olga; Agapkina, Julia; Knyazhanskaya, Ekaterina; Korolev, Sergey; Starodubova, Elizaveta; Viklund, Alecia; Lukashov, Vladimir; Magnani, Mauro; Medstrand, Patrik; Karpov, Vadim; Gottikh, Marina; Isaguliants, Maria

2014-07-01

371

Decoding the language of phosphorylation site dynamics.  

PubMed

Immune defenses depend on the ability of immunoreceptors to recognize foreign antigens and initiate intracellular signaling when a pathogen is detected. Signal initiation requires spatial reorganization of proteins and site-specific receptor phosphorylation, which leads to engagement of feedback loops. This Journal Club discusses recent work using combined experimental and computational approaches to investigate these processes in B cell antigen receptor (BCR) signaling. Specifically, the roles of different kinases in the presence and absence of BCR clustering were evaluated. Results indicated that spleen tyrosine kinase (SYK) can compensate for loss of Src-family kinase activity when receptors are spatially clustered, in part because receptor clustering enables SYK to trigger a positive feedback loop. This study and its implications suggest additional uses for computational models in studies of immunoreceptor signaling and highlight areas where extensions of current methodology are needed to better understand the complexities of biomolecular interactions. PMID:23652201

Chylek, Lily A

2013-05-01

372

The regulation of STIM1 by phosphorylation  

PubMed Central

Calcium ion (Ca2+) concentration plays a key role in cell signaling in eukaryotic cells. At the cellular level, Ca2+ directly participates in such diverse cellular events as adhesion and migration, differentiation, contraction, secretion, synaptic transmission, fertilization, and cell death. As a consequence of these diverse actions, the cytosolic concentration of free Ca2+ is tightly regulated by the coordinated activity of Ca2+ channels, Ca2+ pumps, and Ca2+-binding proteins. Although many of these regulators have been studied in depth, other proteins have been described recently, and naturally far less is known about their contribution to cell physiology. Within this last group of proteins, STIM1 has emerged as a major contributor to Ca2+ signaling by means of its activity as Ca2+ channel regulator. STIM1 is a protein resident mainly, but not exclusively, in the endoplasmic reticulum (ER), and activates a set of plasma membrane Ca2+ channels termed store-operated calcium channels (SOCs) when the concentration of free Ca2+ within the ER drops transiently as a result of Ca2+ release from this compartment. Knowledge regarding the molecular architecture of STIM1 has grown considerably during the last years, and several structural domains within STIM1 have been reported to be required for the specific molecular interactions with other important players in Ca2+ signaling, such as Ca2+ channels and microtubules. Within the modulators of STIM1, phosphorylation has been shown to both activate and inactivate STIM1-dependent Ca2+ entry depending on the cell type, cell cycle phase, and the specific residue that becomes modified. Here we shall review current knowledge regarding the modulation of STIM1 by phosphorylation.

Pozo-Guisado, Eulalia; Martin-Romero, Francisco Javier

2013-01-01

373

Enzymatic synthesis of delta sleep-inducing peptide.  

PubMed

The delta sleep-inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain- or alpha-chymotrypsin-mediated synthesis. Secondary hydrolysis was suppressed by introducing N alpha-protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C-terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high-performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences. PMID:3366553

Sakina, K; Kawazura, K; Morihara, K

1988-02-01

374

Adhesion improvement of lignocellulosic products by enzymatic pre-treatment.  

PubMed

Enzymatic bonding methods, based on laccase or peroxidase enzymes, for lignocellulosic products such as medium-density fiberboard and particleboard are discussed with reference to the increasing costs of presently used petroleum-based adhesives and the health concerns associated with formaldehyde emissions from current composite products. One approach is to improve the self-bonding properties of the particles by oxidation of their surface lignin before they are fabricated into boards. Another method involves using enzymatically pre-treated lignins as adhesives for boards and laminates. The application of this technology to achieve wet strength characteristics in paper is also reviewed. PMID:18502077

Widsten, Petri; Kandelbauer, Andreas

2008-01-01

375

Enzymatic Processing of Bioactive Glycosides from Natural Sources  

NASA Astrophysics Data System (ADS)

A number of biologically active natural products are glycosides. Often, the glycosidic residue is crucial for their activity. In other cases, glycosylation only improves their pharmacokinetic parameters. Enzymatic modification of these glycosides - both extension of the glycoside moiety and its selective trimming - is advantageous due to their selectivity and mildness of the reaction conditions in the presence of reactive and sensitive complex aglycones. Enzymatic reactions enable the resulting products to be used as "natural products", e.g., in nutraceuticals. This chapter concentrates on naturally occurring glycosides used in medicine but also in the food and flavor industry (e.g., sweeteners). Both "classical" and modern methods will be discussed.

Weignerová, Lenka; K?en, Vladimír

376

Protein microchips : use for immunoassay and enzymatic reactions.  

SciTech Connect

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-{mu}m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloschuk, S.; Chupeeva, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences

2000-02-15

377

Phosphorylation-induced Conformational Changes in Rap1b  

PubMed Central

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser179 at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser179 phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

Edreira, Martin M.; Li, Sheng; Hochbaum, Daniel; Wong, Sergio; Gorfe, Alemayehu A.; Ribeiro-Neto, Fernando; Woods, Virgil L.; Altschuler, Daniel L.

2009-01-01

378

RNA splicing: What has phosphorylation got to do with it?  

Microsoft Academic Search

Many pre-mRNA splicing factors are phosphorylated in vivo, but the role of this modification has been unclear. Recent observations suggest that phosphorylation modulates protein–protein interactions within the spliceosome, thereby contributing to dynamic structural reorganization of the spliceosome during splicing.

Tom Misteli

1999-01-01

379

Detection of Intracellular Phosphorylated STAT1 by Flow Cytometry  

Microsoft Academic Search

We have applied flow cytometry to the investigation of interferon-? activation of human monocytes. This approach uses monoclonal antibodies that distinguish between the native and phosphorylated forms of STAT-1. It enables rapid and quantitative assessment of STAT-1 phosphorylation on a discrete cell basis and is both more sensitive and less time consuming than immunoblotting. Furthermore, it allows for discrimination between

Thomas A. Fleisher; Susan E. Dorman; Jill A. Anderson; Michael Vail; Margaret R. Brown; Steven M. Holland

1999-01-01

380

Host Cell Interactome of Tyrosine-Phosphorylated Bacterial Proteins  

Microsoft Academic Search

Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use secretion systems to inject tyrosine kinase substrates into host cells. Upon phosphorylation, these effector proteins recruit cellular binding partners to manipulate host cell functions. So far, only a few interaction partners have been identified. Here we report the

Matthias Selbach; Florian Ernst Paul; Sabine Brandt; Patrick Guye; Oliver Daumke; Steffen Backert; Christoph Dehio; Matthias Mann

2009-01-01

381

Literature mining of protein phosphorylation using dependency parse trees.  

PubMed

As one of the most common post-translational modifications (PTMs), protein phosphorylation plays an important role in various biological processes, such as signaling transduction, cellular metabolism, differentiation, growth, regulation and apoptosis. Protein phosphorylation is of great value not only in illustrating the underlying molecular mechanisms but also in treatment of diseases and design of new drugs. Recently, there is an increasing interest in automatically extracting phosphorylation information from biomedical literatures. However, it still remains a challenging task due to the tremendous volume of literature and circuitous modes of expression for protein phosphorylation. To address this issue, we propose a novel text-mining method for efficiently retrieving and extracting protein phosphorylation information from literature. By employing natural language processing (NLP) technologies, this method transforms each sentence into dependency parse trees that can precisely reflect the intrinsic relationship of phosphorylation-related key words, from which detailed information of substrates, kinases and phosphorylation sites is extracted based on syntactic patterns. Compared with other existing approaches, the proposed method demonstrates significantly improved performance, suggesting it is a powerful bioinformatics approach to retrieving phosphorylation information from a large amount of literature. A web server for the proposed method is freely available at http://bioinformatics.ustc.edu.cn/pptm/. PMID:24440484

Wang, Mang; Xia, Hong; Sun, Dongdong; Chen, Zhaoxiong; Wang, Minghui; Li, Ao

2014-06-01

382

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain.  

PubMed Central

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation. Images

Vassetzky, Y S; Dang, Q; Benedetti, P; Gasser, S M

1994-01-01

383

Tau Phosphorylation and ?-Calpain Activation Mediate the Dexamethasone-Induced Inhibition on the Insulin-Stimulated Akt Phosphorylation  

PubMed Central

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of ?-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and ?-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and ?-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and ?-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated ?-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and ?-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and ?-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.

Liu, Yudong; Su, Ying; Sun, Shenggang; Wang, Tao; Qiao, Xian; Run, Xiaoqin; Liang, Zhihou

2012-01-01

384

Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.  

PubMed

Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization. PMID:24364495

Wiese, Heike; Kuhlmann, Katja; Wiese, Sebastian; Stoepel, Nadine S; Pawlas, Magdalena; Meyer, Helmut E; Stephan, Christian; Eisenacher, Martin; Drepper, Friedel; Warscheid, Bettina

2014-02-01

385

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase  

SciTech Connect

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca/sup 2 +/-ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca/sup 2 +/ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover.

Teruel, J.A.; Inesi, G.

1988-08-09

386

Sprouty2 interacts with protein kinase C delta and disrupts phosphorylation of protein kinase D1.  

PubMed

The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C delta (PKCdelta) and Spry2. Endogenous PKCdelta and Spry2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length Spry2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCdelta by another kinase, it reduced the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCdelta with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCdelta, it was more likely to form a trimeric complex with both PKCdelta and PKD1. Formation of the complex was found to be dependent on an existing PKCdelta-PKD1 interaction. By disrupting the interaction between PKCdelta and PKD1, Spry2 was unable to associate with PKCdelta to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCdelta and PKD1 was increased, and the transfer of phosphate groups from PKCdelta to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCdelta resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCdelta signaling. By disrupting the capacity of PKCdelta to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCdelta signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome. PMID:19458088

Chow, Soah Yee; Yu, Chye Yun; Guy, Graeme R

2009-07-17

387

Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling  

PubMed Central

The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N-terminal domain of Ndc80 (Ndc801?257), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc801?257 in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild-type-like bipolar kinetochore–microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1-dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores.

Kemmler, Stefan; Stach, Manuel; Knapp, Maria; Ortiz, Jennifer; Pfannstiel, Jens; Ruppert, Thomas; Lechner, Johannes

2009-01-01

388

Phosphorylation of tau protein by recombinant GSK-3?: pronounced phosphorylation at select Ser\\/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain  

Microsoft Academic Search

Glycogen synthase kinase-3? (GSK-3?) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3? can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau

R. Godemann; J. Biernat; E. Mandelkow

1999-01-01

389

The participation of calponin in the cross talk between 20-hydroxyecdysone and juvenile hormone signaling pathways by phosphorylation variation.  

PubMed

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to mediate insect development, but the mechanism of this interaction is poorly understood. Here, a calponin homologue domain (Chd) containing protein (HaCal) is reported to play a key role in the cross talk between 20E and JH signaling by varying its phosphorylation. Chd is known as an actin binding domain present in many proteins including some signaling proteins. Using an epidermal cell line (HaEpi), HaCal was found to be up-regulated by either 20E or the JH analog methoprene (JHA). 20E induced rapid phosphorylation of HaCal whereas no phosphorylation occurred with JHA. HaCal could be quickly translocated into the nuclei through 20E or JH signaling but interacted with USP1 only under the mediation of JHA. Knockdown of HaCal by RNAi blocked the 20E inducibility of USP1, PKC and HR3, and also blocked the JHA inducibility of USP1, PKC and JHi. After gene silencing of HaCal by ingestion of dsHaCal expressed by Escherichia coli, the larval development was arrested and the gene expression of USP1, PKC, HR3 and JHi were blocked. These composite data suggest that HaCal plays roles in hormonal signaling by quickly transferring into nucleus to function as a phosphorylated form in the 20E pathway and as a non-phosphorylated form interacting with USP1 in the JH pathway to facilitate 20E or JH signaling cascade, in short, by switching its phosphorylation status to regulate insect development. PMID:21625546

Liu, Peng-Cheng; Wang, Jin-Xing; Song, Qi-Sheng; Zhao, Xiao-Fan

2011-01-01

390

Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination.  

PubMed

Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr(20)) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr(27). Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr(20) caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr(27) mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr(20) is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXX? (where X is any amino acid and ? is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr(20) both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr(20) may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

Chesarino, Nicholas M; McMichael, Temet M; Hach, Jocelyn C; Yount, Jacob S

2014-04-25

391

Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of C3 Plants Undergoes Light-/Dark-Induced Reversible Phosphorylation1  

PubMed Central

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C4 cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C3 plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C3 PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C4 dikinase. In addition, the kinetics of this process closely resemble the reversible C4 process, with light-induced dephosphorylation occurring rapidly (?15 min) and dark-induced phosphorylation occurring much more slowly (?30–60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C3 PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C3-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C3 plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C4 pathway regulatory “converter” enzyme.

Chastain, Chris J.; Fries, Jason P.; Vogel, Julie A.; Randklev, Christa L.; Vossen, Adam P.; Dittmer, Sharon K.; Watkins, Erin E.; Fiedler, Lucas J.; Wacker, Sarah A.; Meinhover, Katherine C.; Sarath, Gautam; Chollet, Raymond

2002-01-01