Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Divalent cations stabilized rat recombinant O6-methylguanine-DNA methyltransferase (rMGMT) protein against heat treatment. Activity of rMGMT was completely abolished by incubating at 45 degrees C for 30 min, however, addition of 1.0 mM Mg2+, Ca2+ or Mn2+ significantly protected heat-induced inactivation of MGMT activity (50-60% vs. 97% inactivation). Protective effect of Ca2+ on the MGMT activity was concentration-dependent up to 3 mM, and the thermal protection was effective up to 45 degrees C. In order to investigate Ca2+ binding site in rMGMT protein, truncated GST-rMGMT proteins containing N-terminal 39 amino acids (GST-rMGMT39), 70 amino acids (GST-rMGMT70) and full-length protein (GST-rMGMT) were prepared. Radiolabeled calcium ion [45Ca2+] was bound only to the GST-rMGMT70 and GST-rMGMT, but not to the GST-rMGMT39, indicating that divalent cations could bind the residues between 40th and 70th of the rMGMT protein. Calcium binding was not observed in the site-directed mutant rMGMT proteins (rMGMT(D42A) and rMGMT(E45A)), confirmed by autoradiography using [45Ca2+] after nondenaturing gel electrophoresis; however, the above two mutants had the same catalytic activity as well as proteolytic sensitivity as the wild MGMT protein. Analysis by equilibrium dialysis revealed stoichiometric binding of one molecule of Ca2+ to one molecule of the protein. Since circular dichroism (CD) spectra indicated no discernible difference before and after Ca2+ binding, the above results suggested that neutralization of two negative charges of Asp42 and Glu45 by Ca2+ resulted in thermal stabilization of the protein with minimum perturbation of its tertiary structure.
 ...
PMID:Induction of thermal and chemical stability of O6-methylguanine-DNA methyltransferase by Ca2+. 1247 3

Many individuals with cardiovascular diseases undergo physical conditioning with or without medication. Therefore, this study investigated the interaction of exercise training and chronic nitroglycerin treatment on blood pressure (BP) and changes in cardiac nitric oxide (NO) and antioxidants in rats. Fisher 344 rats were divided into four groups treated as: (1) sedentary control, (2) exercise training for 8 weeks, (3) nitroglycerin (15 mg/kg, s.c. for 8 weeks), and (4) training+nitroglycerin for 8 weeks. Respiratory exchange ratio (RER), BP, and heart rate (HR) were monitored weekly for 8 weeks. The animals were sacrificed 24 h after last treatments, hearts isolated, and analyzed. Physical conditioning significantly increased RER, cardiac NO levels, and endothelial eNOS protein expression. Training significantly enhanced cardiac glutathione (GSH) levels, GSH/GSSG ratio, and the up-regulation of cardiac copper/zinc-superoxide dismutase (CuZn-SOD), manganese (Mn)-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) activities, and protein expression. Training also caused depletion of cardiac malondialdehyde (MDA) and protein carbonyls with a significant increase in RER without any change in BP and HR. Chronic nitroglycerin administration significantly increased cardiac NO levels and eNOS protein expression. Nitroglycerin administration significantly enhanced cardiac Mn-SOD, CAT, and GST activities, and protein expression with decreased MDA levels and BP. Interaction of training and chronic nitroglycerin treatment increased cardiac NO levels with enhanced eNOS and iNOS protein expressions, GSH/GSSG ratio, and the up-regulation of antioxidant enzymes. This interaction normalized BP and HR and increased RER. The data suggest that the interaction of physical training and chronic nitroglycerin treatment resulted in the maintenance of BP and RER by up-regulating the antioxidants and NO levels and by reducing the oxidative stress in the rat heart.
 ...
PMID:Interaction of physical training and chronic nitroglycerin treatment on blood pressure, nitric oxide, and oxidants/antioxidants in the rat heart. 1286 Apr 43

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.
 ...
PMID:Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein. 1286 Sep 96

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.
 ...
PMID:Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase. 1455 Jun 51

We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent K(m) of approximately 0.4 microM. The apparent K(m) for ATP was determined to be approximately 3 microM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the K(m) concentrations of the two substrates for at least 2 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC(50) of roughly 0.8 microM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4 degrees C storage was seen out to 6 h with an approximately 50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of approximately 24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.
 ...
PMID:Characterization of the kinase domain of the ephrin-B3 receptor tyrosine kinase using a scintillation proximity assay. 1509 Feb 52

The effect of cadmium or manganese administration on rat liver glutathione S-transferase (GST) has been investigated. The activity of this enzyme in liver cytosol, where almost all the cellular activity is present, had increased by more than 36% 24 h after a single i.p. injection of CdCl(2) (2.5 mg kg(-1) b.w.) or MnCl(2) (2.0 mg kg(-1) b.w.). After shorter and longer time intervals, a lower enzyme activity stimulation was observed in both cases. When liver cytosol was incubated for 10 min with 75 microM CdCl(2) or 40 microM MnCl(2), no effect was observed on enzyme activity. The increase in GST following cadmium or manganese administration was blocked by prior administration of actinomycin D, indicative of a possible transcription-dependent response. The liver soluble GST from both control and metal-treated rats was not at all affected by Vitamin E, in the range of 20-300 microM. By contrast, hematin was seen to be a competitive inhibitor of this liver enzyme from both types of rats by using CDNB as substrate and the K(i) value was equal to 0.22 microM. The possibility that under the conditions used class alpha GST isoenzymes are affected by cadmium or manganese is discussed.
 ...
PMID:Rat liver glutathione S-transferase activity stimulation following acute cadmium or manganese intoxication. 1515 61

The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date.
 ...
PMID:ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation. 1531 75

The activities of copper, zinc-superoxide dismutase (SOD1), manganese SOD (SOD2), glutathione peroxidase (GPX), glutathione reductase (GSSG-R) and glutathione S-transferase (GST) were studied in sheep corpora lutea (CL) obtained on days 15, 40, 60, 80 and 128 of pregnancy. Maintained enzymatic activity of SOD1, SOD2, GPX, GSSG-R and GST were found in the sheep CL throughout pregnancy. Enzymatic activity of SOD1, GPX and GST increased significantly from day 15 to day 40 of pregnancy, and thereafter remained constant until day 128. SOD2 and GSSG-R activities were not different between any days of pregnancy examined. Apoptotic luteal cells identified by the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling were very rarely observed, and their incidence (less than 0.5%) was not different between days of pregnancy. These results showed that the activities of antioxidant enzymes in the sheep CL are subject to major changes during early pregnancy, suggesting that the CL of early pregnancy may be rescued from luteolysis through increasing activities of key antioxidant enzymes and inhibition of apoptosis. Maintained levels of antioxidant enzymes in the CL throughout pregnancy may be linked to reactive oxygen species continuously generated in the steroidogenically active luteal cells, and may be involved in the maintenance of luteal steroidogenic activity and cellular integrity.
 ...
PMID:Antioxidant enzymatic defence systems in sheep corpus luteum throughout pregnancy. 1557 94

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.
 ...
PMID:In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis. 1604 3

Three similar but mechanistically distinct fosfomycin resistance proteins that catalyze the opening of the oxirane ring of the antibiotic are known. FosA is a Mn(II) and K(+)-dependent glutathione transferase. FosB is a Mg(2+)-dependent L-cysteine thiol transferase. FosX is a Mn(II)-dependent fosfomycin-specific epoxide hydrolase. The expression, purification, kinetic, and physical characteristics of six fosfomycin resistance proteins including the FosA proteins from transposon TN2921 and Pseudomonas aeruginosa, the FosB proteins from Bacillus subtilis and Staphylococcus aureus, and the FosX proteins from Mesorhizobium loti and Listeria monocytogenes are reported.
 ...
PMID:Fosfomycin resistance proteins: a nexus of glutathione transferases and epoxide hydrolases in a metalloenzyme superfamily. 1639 98


<< Previous 1 2 3 4 5 6 7 8 9 Next >>