Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.
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PMID:Rate-determining steps for tyrosine phosphorylation by the kinase domain of v-fps. 863 84

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).
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PMID:The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae. 864 87

Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
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PMID:Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. 866 21

Metabolism of methylene chloride, or dichloromethane (DCM), plays a key role in determining the kinetics and carcinogenicity of the halocarbon. The objectives of this study were: to evaluate and optimize the vial equilibration technique, originally described by Sato and Nakajima (1979a), in order to characterize the hepatic metabolism of DCM by Sprague-Dawley rats; to employ different hepatic microsomal preparations to examine buffer effects on DCM metabolism; and to assess the relative importance and metabolic constants of the mixed-function oxidase (MFO) and glutathione (GSH) S-transferase (GST) metabolic pathways. A crude liver homogenate (20% W/V) was prepared from perfused livers of male Sprague-Dawley (S-D) rats (275-325 g). A 30% glycerol buffer was found to significantly inhibit DCM metabolism, while 0.25 M sucrose buffer containing 10 mM EDTA and 1.15% KCl did not. DCM was incubated with the liver 10,000 g supernatant or microsomes and cofactors in sealed headspace vials. Disappearance of DCM, as a measure of the chemical's metabolism, was monitored by headspace gas chromatography. Different trials were conducted to elucidate time-, enzyme-, and substrate-activity relationships. The scaled-up K(m) and Vmax values for the microsomal fraction were quite similar to optimized in vivo values reported by other investigators. In the current study, DCM appeared to be metabolized preferentially by cytochrome P450 IIE1, since substrates (e.g., pyrazole, ethanol, and glycerol) for this isozyme completely inhibited DCM metabolism. Thus, glycerol should not be used as a P450 stabilizer for preparation or storage of microsomes. Phorone pretreatment caused marked hepatic GSH depletion, but had little effect on the overall rate of DCM metabolism. Quantitatively, the GST pathway in the cytosol played a very minor role in DCM metabolism. It was not possible to accurately calculate metabolic constants for this pathway in S-D rats. The vial equilibration technique, as described here, is a relatively simple and reliable method, which should be broadly applicable for measuring the microsomal metabolism of DCM and other VOCs.
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PMID:Use of the vial equilibration technique for determination of metabolic rate constants for dichloromethane. 880 40

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.
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PMID:Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae. 1004 86

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX-AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST-AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX-AfsK-g in the presence of [gamma-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX-AfsK-g phosphorylated serine and threonine residues of GST-AfsR-g in the presence of [gamma-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism.
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PMID:An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus. 1051 81

We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
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PMID:Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli. 1096 39

Phosphorylation of eIF4E is required for protein synthesis during starfish oocyte maturation. The activity of protein kinase C-related kinase 2 (PRK2) increases prior to the phosphorylation of eIF4E (G. Stapleton et al., 1998, Dev. Biol. 193, 34-46). We investigate here whether eIF4E is activated by PRK2. A 3.5-kb eIF4E clone isolated from starfish cDNA is 57% identical to human eIF4E and contains the putative phosphorylation site serine-209. The serine-209 environment (SKTGS(209)MAKSRF) is similar to the consensus sequence of the phosphorylation site of protein kinase C and related kinases. A starfish eIF4E fusion protein (GST-4E) was phosphorylated in vitro by PRK2 in the presence of 1,2-diolyl-sn-glycerol 3-phosphate. In contrast, replacing the GST-4E serine-209 with an alanine significantly reduced this phosphorylation. Analysis by two-dimensional phosphopeptide mapping reveals a major phosphopeptide in trypsin-digested GST-4E, but not in its serine-209 mutant. Importantly, this major phosphopeptide in GST-4E corresponds to a major phosphopeptide of eIF4E isolated from (32)P-labeled oocytes. Thus, PRK2 may regulate translation initiation during oocyte maturation by phosphorylating the serine-209 residue of eIF4E in starfish. We also demonstrate that high levels of cAMP inhibit the activation of PRK2, eIF4E, and the eIF4E binding protein during starfish oocyte maturation, while PI3 kinase activates these proteins.
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PMID:Protein kinase C-related kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes. 1111 22

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.
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PMID:A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment. 1126 67

Plant elongation factor EF-1 consists of four subunits (EF-1alphabetabeta'gamma). EF-1alpha. GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1beta and EF-1beta' catalyze the GDP/GTP exchange on EF-1alpha. GDP. However, the function of EF-1gamma, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1betabeta'gamma and recombinant EF-1gamma possess glutathione S-transferase (GST) activity. The EF-1betabeta'gamma- or EF-1gamma-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1betabeta'gamma, EF-1gamma, and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1gamma is heat labile, active EF-1gamma was obtained by purifying it in the presence of 20% glycerol.
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PMID:Detection and characterization of glutathione S-transferase activity in rice EF-1betabeta'gamma and EF-1gamma expressed in Escherichia coli. 1167 72


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