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)

For all neoplasms, extraneural as well as brain, intrinsic, and acquired resistance to antineoplastic drugs constitutes a multifactorial problem. Much information has been generated concerning the individual mechanisms that play a role in drug resistance. The present decade will see a great deal of laboratory research emphasis in two related areas: (1) the molecular biology of resistance, including processes that regulate gene expression for critical detoxifying and transport proteins, and (2) further identification of DNA repair mechanisms in normal and neoplastic cells. In addition to continued research directed toward the identification of specific mechanisms, further study of the interrelationship between these mechanisms will be essential. Finally, there is a growing awareness that in vitro determination of the rank order of mechanisms contributing to resistance for a given drug may be quite different from that determined in vivo. The complexity of this problem is increased for brain tumors in that the understanding of the fundamentals of brain tumor biology is less advanced than for many of the systemic tumors. Ultimately, the identification of resistance mechanisms will lead to the development of clinically useful approaches to reverse cellular resistance and to increase drug sensitivity. Examples of such strategies that have or will find their way into clinical trial include: (1) use of buthionine sulfoximine to reverse glutathione-mediated resistance, (2) use of ethacrynic acid to reverse glutathione S-transferase-mediated resistance, and (3) use of calcium channel blockers and calmodulin inhibitors to reverse MDR. There will also be considerable emphasis on the rational modification of existing antineoplastic agents and the development of new drugs designed to circumvent important resistance mechanisms. For brain tumor treatment, additional strategies to circumvent intrinsic and acquired resistance by increasing drug delivery, such as high-dose chemotherapy with marrow or growth factor rescue and local drug delivery to brain tumors by drug-impregnated biodegradable polymers, will continue to be examined. Previous experience with efforts to augment antineoplastic drug cytotoxicity indicates that this process may decrease the margin of cytotoxicity between normal tissue and tumor, often referred to as the therapeutic index. To avoid serious neurotoxicity as a dose-limiting or treatment-limiting factor for potentially important clinical strategies to modulate drug resistance, it will be important to develop a greater understanding of the relative treatment sensitivities of brain capillary endothelium, glial cells, and neurons, as well as their individual abilities to transport, detoxify, and repair the effects of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Antineoplastic drug resistance in brain tumors. 168 94

Stress, catecholamines (CA), cAMP and protein-kinase A do not affect superoxide dismutase, catalase, thioredoxin reductase, thiol transferase and glutathione reductase (GR). However, they activate glutathione peroxidase and glutathione transferase (GT) in a number of organs and inhibit renal gamma-glutamyl transferase. Ca2+ ions activate GT through calmodulin. CA were found to stimulate GSH transport from liver to blood and GT phosphorylation by protein kinase C. This suggests a regulation of the GSH metabolism by hormones and a second messenger. This regulation favours metabolism of active O2 substances (including protection from peroxide stress and leukotriene C4 synthesis), supporting of SH-proteins in reduced state, xenobiotics detoxication. GT and GR induction can play an important role in the mechanism of anti-peroxide action of butylhydroxytoluene.
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PMID:[The physiological significance of regulation by catecholamines, second messengers and enzyme inducers of glutathione metabolism]. 196 98

This report describes the isolation of the major calmodulin-stimulated methyl acceptor protein of adult rat liver cytosol. This Mr 29,000 methyl acceptor protein (MeAP29) has been purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and Sephadex G-75. Affinity chromatography on glutathione-Sepharose and assays of enzyme activity indicate that MeAP29 is a member of the glutathione S-transferase family. We further show that glutathione can act as an inhibitor of calmodulin-stimulated in vitro methylation of MeAP29 and that MeAP29 methylation is enhanced in non-dialyzed liver cytosol from rats with lowered glutathione levels.
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PMID:Identification of glutathione S-transferase as a substrate and glutathione as an inhibitor of in vitro calmodulin-stimulated protein methylation in rat liver cytosol. 317 41

The mechanism for the regulation of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was investigated using a series of COOH-terminal truncated mutants. These mutants were expressed in bacteria as fusion proteins with glutathione S-transferase and purified by affinity chromatography using glutathione Sepharose 4B. A mutant (residues 1-332) showed complete Ca2+/CaM-dependent activity. Truncation mutants (residues 1-321, 1-314, and 1-309) exhibited decreasing affinities for Ca2+/CaM and also exhibited decreasing Ca2+/CaM-dependent activities. Truncation mutants (residues 1-305 or 1-299) were unable to bind Ca2+/CaM and were inactive. In contrast, truncation mutants (residues 1-293 or 1-277) were constitutively active at a slightly higher level (2-fold) than fully active CaM kinase I. These results indicate the location of the Ca2+/CaM-binding domain on CaM kinase I (residues 294-321) and predict the existence of an autoinhibitory domain near, or overlapping, the Ca2+/CaM-binding domain. These conclusions were supported by studies which showed that a synthetic peptide (CaM kinase I (294-321)) corresponding to residues 294-321 of CaM kinase I inhibited the fully active kinase in a manner that was competitive with Ca2+/CaM and also inhibited the constitutively active mutant (residues 1-293) in a manner that was competitive with Syntide-2, a peptide substrate, (Ki = 1.2 microM) but was non-competitive with ATP. Thus, these results suggest that CaM kinase I is regulated through an intrasteric mechanism common to other members of the family of Ca2+/CaM-dependent protein kinases.
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PMID:The regulatory region of calcium/calmodulin-dependent protein kinase I contains closely associated autoinhibitory and calmodulin-binding domains. 755 63

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.
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PMID:Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli. 772 24

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17

We have cloned a full-length cDNA from Dictyostelium discoideum which encodes a new Ca(2+)-binding protein. The deduced protein (termed CBP1) is composed of 156 amino acids and contains four consensus metal-ligating loop sequences found in helix-loop-helix motifs of many Ca(2+)-binding proteins. When expressed in bacteria as a GST fusion protein, CBP1 binds Ca2+ in a 45Ca2+ overlay assay. CBP1 exhibits little amino acid sequence homology with Dictyostelium calmodulin or calfumirin-1 (CAF-1) except in the putative Ca(2+)-binding regions. Moreover, unlike calmodulin and CAF-1 expression, CBP1 mRNA is expressed preferentially during the multicellular stages of development.
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PMID:Cloning and expression in Escherichia coli of a cDNA encoding a developmentally regulated Ca(2+)-binding protein from Dictyostelium discoideum. 772 26

Calcium-dependent protein kinases (CDPKs) represent a new family of protein kinases which are proposed to contain, in a single polypeptide, both a kinase domain and an adjoining calmodulin-like domain with four calcium-binding EF-hand motifs [Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., & Harmon, A.C. (1991) Science 252, 951-954]. DNA cloning and Western blot analysis indicate that multiple CDPK isoforms are present in the model plant system Arabidopsis thaliana. One CDPK gene called AK1 was isolated from Arabidopsis as a full-length cDNA. The predicted AK1 protein has a M(r) of 72,645 and is 116 amino acid residues longer at the amino terminus than the prototype CDPK alpha gene previously identified in soybean. The most highly conserved region between these two CDPKs is a region of 31 amino acids that joins the kinase and calmodulin-like domains. To verify the kinase activity of the enzyme encoded by AK1, a fusion of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase was expressed in Escherichia coli. The fusion protein was purified and displayed a maximum kinase activity of 40 nmol of phosphate/(min.mg), using histone IIIs as a substrate. The enzyme activity was stimulated 3-6-fold by calcium and 2-5-fold by crude lipid. However, a synergistic stimulation of 16-30-fold was observed by the addition of both calcium and crude lipid. Lipid stimulation was specific for lysophosphatidylcholine and phosphatidylinositol and did not occur with the addition of phosphatidylserine or phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli. 791 21

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.
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PMID:Expression and characterization of a rac family protein kinase of Entamoeba histolytica. 798 73

CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.
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PMID:Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain. 800 90


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