On the discovery of glutathione.
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Glutathione↗
Biomedical subjects
A Meister
Publications and source records attributed to A Meister.
On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.
gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group. Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate. However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol. Thus, the rat kidney and E. coli enzymes share several catalytic features but differ in active site structure. If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.
Animals↗
[A computer program for the quantitative evaluation of enzyme immunoassay data].
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Computer Simulation↗
[Reaction of the cardiovascular system, temporary threshold shift 2 and attitude in local dynamic muscle work with noise].
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Adult↗
Subjective evaluation of isolated and combined exposure to whole-body vibration and noise by means of cross-modality matching.
Cross-modality matching (CMM) were used for the evaluation of combined effects of sinusoidal whole-body vibration (WBV) in Z-axis with the frequencies 3-7 Hz, 1.5 ms-2, and two noise levels (L1 = 65 dBA; L2 = 85 dBA). CMM's were tested by means of three responses: handgrip force, length, estimation and brightness adjustment. The subjects were instructed to set different modalities to a value corresponding to their perception of vibration. The combination of WBV and L2 caused higher sensation responses than WBV and L1.
Analysis of Variance↗
Inactivation of gamma-glutamylcysteine synthetase, but not of glutamine synthetase, by S-sulfocysteine and S-sulfohomocysteine.
The reactions catalyzed by gamma-glutamylcysteine synthetase and glutamine synthetase are thought to proceed via enzyme-bound gamma-glutamyl phosphate intermediates. We investigated the possibility that S-sulfocysteine and S-sulfohomocysteine might act as analogs of gamma-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D- and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney gamma-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of gamma-glutamylcysteine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inactivator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme.
Animals↗
18O studies on the 5-oxoprolinase reaction. Evidence for a phosphorylated tetrahedral intermediate.
5-Oxoprolinase catalyzes a reaction in which the cleavage of ATP to ADP and Pi and the decyclization of 5-oxoproline to form glutamate are coupled. When the reaction catalyzed by 5-oxoprolinase of Pseudomonas putida was carried out to 90% completion in H2(18)O, the residual 5-oxoproline was found to contain 18O in the amide carbonyl oxygen atom. Such isotopic incorporation was not observed in similar studies with a subunit of the enzyme which catalyzes 5-oxoproline-dependent ATPase and formation of a phosphorylated 5-oxoproline intermediate (Seddon, A.P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11543). When the complete reaction was carried out in H2(18)O, the products glutamate (gamma-carboxyl) and inorganic phosphate were mono- and di-labeled with 18O. Studies with 5-[18O]oxo-L-proline confirmed such replacement of the oxygen atoms of the gamma-carboxyl group of glutamate and the carbonyl oxygen of 5-oxoproline. Oxygen was not transferred from 5-oxoproline to inorganic phosphate. Studies with analogs of 5-oxoproline showed that di-labeling of inorganic phosphate occurred only when ATP hydrolysis was coupled or partially coupled with the decyclization of the substrate. Studies with 5-oxoprolinase from rat kidney gave similar results. These observations are in accord with the view that the reaction involves enzyme-bound phosphorylated intermediates and provide evidence for a phosphorylated tetrahedral intermediate, whose formation is required for coupling.
1-Carboxyglutamic Acid↗
Fluorometric determination of alpha-ketosuccinamic acid in rat tissues.
A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.
Animals↗
Enzymatic synthesis of novel glutathione analogs.
A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K. Murata, W. A. Abbott, R. J. Bridges, and A. Meister (1985) Anal. Biochem. 150, 235-237). In the present work, this E. coli matrix was used as the basis of a method for the synthesis of glutathione analogs. Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures. Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine. This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids. The analogs are purified by gel-filtration and ion-exchange chromatography. The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function. Fluorine-containing analogs may be used for NMR studies. The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.
Amino Acids↗
Intracellular delivery of cysteine.
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Animals↗
Glutathione, a first line of defense against cadmium toxicity.
Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.
1-Propanol↗
Interaction of the D-isomer of gamma-methylene glutamate with an active site thiol of gamma-glutamylcysteine synthetase.
gamma-Glutamylcysteine synthetase has a thiol group in the vicinity of its glutamate-binding site. During efforts to find a covalently bound inhibitor, interaction of the enzyme with gamma-methylene glutamate was examined because this analog of glutamate, which has an alpha,beta-unsaturated moiety, would be expected to bind at the glutamate site and might react with an active site thiol. gamma-Methylene glutamate, which is not a significant substrate, inhibits the enzyme competitively toward glutamate. Preincubation of the enzyme with gamma-methylene DL-glutamate led to substantial inactivation which was dependent upon the presence of Mg2+ or Mn2+; glutamate protected against inactivation. Inactivation was observed with the D-isomer of gamma-methylene glutamate, but not with the corresponding L-isomer. The inactivated enzyme contains close to 1 mol of gamma-methylene glutamate/mol of enzyme. Studies in which enzyme inactivated by treatment with [14C]gamma-methylene glutamate was hydrolyzed indicate that gamma-methylene glutamate reacts with an active site thiol.
Animals↗
Gamma-glutamyl-glutathione. Natural occurrence and enzymology.
The natural occurrence of gamma-glutamyl-glutathione (gamma-glutamyl-gamma-glutamylcysteinylglycine) in bile was established by analytical and chromatographic studies on the isolated and chemically synthesized materials. Evidence that it is formed in kidney was obtained. The origin of gamma-glutamyl-glutathione was explored through studies on the interaction of glutathione with gamma-glutamyl transpeptidase. When purified gamma-glutamyl transpeptidase was incubated with various concentrations (4 microM-50 mM) of glutathione, the initial rates of formation of gamma-glutamyl-glutathione were substantial at all concentrations of glutathione studied and were greater than the rates of formation of glutamate at physiological levels of glutathione (1-10 mM). The findings indicate that gamma-glutamyl transpeptidase catalyzes transpeptidation in vivo. That gamma-glutamyl-glutathione is formed in vivo and that it is a significant product of the reaction between glutathione and gamma-glutamyl transpeptidase under physiological conditions suggest that this polyanionic tetrapeptide may have a physiological role. gamma-Glutamyl-glutathione is not a substrate of glutathione reductase or of glutathione S-transferase, but it is a substrate of gamma-glutamyl-cyclotransferase. That gamma-glutamyl-glutathione has an additional negative charge as compared to glutathione suggests that it may be more effective than glutathione in forming complexes with certain metal ions and other cations.
Acyltransferases↗
Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase.
Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.
Adenosine Triphosphate↗
Inhibition of gamma-glutamyl transpeptidase and induction of glutathionuria by gamma-glutamyl amino acids.
Treatment of mice with various gamma-glutamyl amino acids leads to marked urinary excretion of glutathione and other gamma-glutamyl compounds. There is good correlation between the affinity of gamma-glutamyl transpeptidase for various gamma-glutamyl amino acids and the extent of glutathionuria. The findings indicate that the administered gamma-glutamyl compounds effectively compete with glutathione (exported from kidney cells and present in the glomerular filtrate) for the enzyme. The administration of certain gamma-glutamyl amino acids appears to be a specific and nontoxic procedure for in vivo inhibition of gamma-glutamyl transpeptidase that may be useful in experimental work on glutathione metabolism and function and also for treatment of certain toxicities and for modulation of the metabolism of endogenous glutathione conjugates.
Amino Acids↗
Intrahepatic transport and utilization of biliary glutathione and its metabolites.
Glutathione transported by hepatocytes into the bile canaliculi is metabolized by the actions of gamma-glutamyl transpeptidase and dipeptidase located on the biliary ductular epithelium. This pathway is revealed by the finding of high levels of cyst(e)inylglycine, gamma-glutamylglutathione, gamma-glutamylcyst(e)ine, glutamate, glycine, and cyst(e)ine in bile, by studies in which intrahepatic metabolism of glutathione was inhibited by administration of a potent inhibitor of gamma-glutamyl transpeptidase and by experiments in which glutathione synthesis was inhibited. Canalicular transport of glutathione, as estimated from totals of metabolites found, is much greater than the glutathione found in bile. Glutathione and glutathione metabolites found in bile increase with age, in association with an increase in hepatic glutathione. In younger rats there is apparent uptake of cysteine and glycine moieties that may reflect uptake of cysteinylglycine at the ductular level. This intrahepatic pathway of glutathione transport and metabolism, which resembles that which occurs in the kidney, seems to function as a cellular protective mechanism in the processing of glutathione conjugates and as a recovery system for cysteine moieties.
Age Factors↗
Intracellular cysteine and glutathione delivery systems.
Glutathione functions in catalysis, metabolism, transport, and reductive processes and in protection of cells by destruction of free radicals, reactive oxygen intermediates, and other toxic compounds of endogenous and exogenous origin. It also functions as a storage and transport form of cysteine. Depletion of glutathione (effectively accomplished by inhibition of its synthesis) increases sensitivity to radiation and to certain toxic compounds and is of value in combination with radiation therapy or chemotherapy in situations in which cell selectivity can be achieved. Increased cellular levels of glutathione protect cells against radiation and certain toxic compounds. Glutathione levels can be increased by administration of cysteine or of glutathione, but these approaches are not entirely satisfactory. Cellular glutathione levels can be increased by supplying substrate for gamma-glutamylcysteine synthetase or for glutathione synthetase. L-2-Oxothiazolidine-4-carboxylate is well transported into many cells and is converted by 5-oxoprolinase to cysteine, a substrate of gamma-glutamylcysteine synthetase. gamma-Glutamylcysteine and related compounds are effectively transported, especially into renal cells, thus providing substrate for glutathione synthetase; higher than normal levels of glutathione can be achieved because this enzyme is not significantly inhibited by glutathione, whereas gamma-glutamylcysteine synthetase is feedback-inhibited. Derivatives of glutathione that are effectively transported into cells (glutathione itself is not) offer another means of increasing glutathione levels. The monoethyl ester of glutathione (in which the glycine carboxyl group is esterified) is well transported in vivo into liver and kidney and into cultured fibroblasts and lymphoid cells. Glutathione levels much higher than usual can be obtained by this procedure, which protects lymphoid cells against the lethal effects of irradiation and mice against acetaminophen, and which therefore may be a relatively safe way to increase cellular resistance to radiation and certain toxic compounds.
Acetaminophen↗
Biological activities and receptor binding of two human recombinant interferons and their hybrids.
Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.
2',5'-Oligoadenylate Synthetase↗