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Biomedical subjects

O W Griffith

Publications and source records attributed to O W Griffith.

At least 127 records · Page 7Linked to original sources
PubMed1980

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism.

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.

Animals↗
PubMed1980

Excretion of cysteine and gamma-glutamylcysteine moieties in human and experimental animal gamma-glutamyl transpeptidase deficiency.

Animals treated with potent gamma-glutamyl transpeptidase inhibitors and a patient with severe gamma-glutamyl transpeptidase deficiency excrete much larger than normal amounts of glutathione, gamma-glutamylcysteine, and cysteine in their urine; these compounds were found in disulfide forms. The findings indicate that the metabolic function of gamma-glutamyl transpeptidase is associated with the metabolism or transport (or both) of cysteine, gamma-glutamylcysteine, and glutathione, and that gamma-glutamylcysteine is a physiological substrate of the enzyme. The occurrence of gamma-glutamylcysteine in urine and other considerations suggest that this dipeptide is formed as an extracellular metabolite of glutathione in addition to its recognized role as an intrcellular precursor of glutathione. The dipeptide may be formed by a pathway involving transpeptidation or by cleavage of the Cys-Gly bond of glutathione. In the course of this work it was found that the mixed disulfide between glutathione and gamma-glutamylcysteine is a good substrate of glutathione reductase.

Animals↗
PubMed1979

Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine).

Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.

Glutamate-Cysteine Ligase↗
PubMed1979

New aspects of glutathione metabolism and translocation in mammals.

An appreciable fraction of the sulphur present in the mammal occurs in the form of glutathione, whose concentration in various tissues ranges from about 0.8 to about 8 mM; the extracellular concentration of glutatione (largely present as the disulphide) is in the micromolecular range. The synthesis of glutathione and its utilization take place by the reactions of the gamma-glutamyl cycle, which include those catalysed by gamma-glutamylcysteine and glutathione synthetases, gamma-glutamyl transpeptidase, cysteinylglycinase, gamma-glutamyl cyclotransferease, and 5-oxoprolinase. gamma-Glutamyl transpeptidase catalyses transpeptidation (with amino acids and dipeptides) and hydrolysis reactions with both blutathione and its disulphide. The transpeptidase is membrane-boudn, apparently to the outer surface of the cell, and is found in certain epithelial cells in anatomical sites that are involved in transport and secretory activities (e.g., renal tubule, jejunal villi, choroid plexus, ciliary body). Evidence that the reactions of the gamma-glutamyl cycle take place in vivo has come from studies with labelled metabolites and selective enzyme inhibitors, and on inborn errors of metabolism associated with specific enzyme deficiencies. Inhibition in vivo of gamma-glutamyl cyclotransferase and 5-oxoprolinase leads, respectively, to decreased and increased renal levels of 5-oxoproline. Administration of a specific inhibitor of gamma-glutamylcysteine synthetase, such as buthionine sulphoximine, leads to a rapid decline in the glutamylcysteine synthetase, such as buthionine sulphoximine, leads to a rapid decline in the glutathione level of the kidney and other tissues, reflecting the appreciable rate of glutathione utilization. When gamma-glutamyl transpeptidase is inhibited in vivo by injection of L- or D-gamma-glutamyl-(o-carboxy)phenylhydrazide, there is extensive glutathionuria and the blood plasma level of glutathione increases. Studies in which inhibitors of glutathione synthesis and transpeptidation were given to mice showed that transport of intracellular glutathione to membrane-bound transpeptidase is a discrete step in the gamma-glutamyl cycle, and that the level of plasma glutatione reflects (a) synthesis of glutathione and its export by liver, muscle, and other tissues and (b) utilization of glutatione by kidney and other tissues. Studies on several lymphoid cell lines show that these cells also actively translocate glutathione out of the cell. A summary scheme is given for the metabolism of glutathione in which glutathione is translocated to the cell membrane where it may be utilized as such or oxidized to glutathione disulphide. Oxidation is inhibited, and transpeptidation is promoted by the presence of amino acids that are substrates of the transpeptidase. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell transport of gamma-glutamyl amino acids and free amino acids...

Animals↗
PubMed1979

Translocation of intracellular glutathione to membrane-bound gamma-glutamyl transpeptidase as a discrete step in the gamma-glutamyl cycle: glutathionuria after inhibition of transpeptidase.

Several inhibitors of gamma-glutamyl transpeptidase in vitro [L-serine plus borate, 6-diazo-5-oxo-L-norleucine, and L- and D-gamma-glutamyl-(o-carboxy)phenylhydrazide] are active in vivo, as indicated by their effect in decreasing the conversion of administered D-gamma-glutamyl-L-alpha-amino[(14)C]butyrate to respiratory (14)CO(2) in mice. The hydrazides (both L and D isomers) are the most potent inhibitors in vitro and in vivo. Inhibition of gamma-glutamyl transpeptidase in vivo by the hydrazides is accompanied by extensive glutahionuria. The evidence suggests that a substantial fraction of the urinary glutathione arises from the kidney. The findings support the view that renal intracellular glutathione is normally translocated to the membrane-bound gamma-glutamyl transpeptidase as a separate step in the gamma-glutamyl cycle. Studies on in vivo inhibition of glutathione synthesis and of gamma-glutamyl transpeptidase provide direct evidence that glutathione is normally translocated from tissues to the blood plasma and that the turnover of plasma glutathione is relatively high. The data suggest that the low but significant steady-state level of glutathione in the plasma reflects synthesis of glutathione (predominantly in the liver) and its utilization by gamma-glutamyl transpeptidase (predominantly in the kidney). Thus, glutathione synthesized in cells that have transpeptidase may be translocated to and used by the membrane-bound enzyme, whereas glutathione synthesized in cells that lack the transpeptidase may be transported via the plasma to transpeptidase located on the membranes of other cells.

Animals↗
PubMed1979

Glutathione: interorgan translocation, turnover, and metabolism.

Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one-third of the total blood plasma glutathione. Normal animals treated with the transpeptidase inhibitor excrete large amounts of glutathione in their urine. They also excrete gamma-glutamylcysteine, suggesting that cleavage of glutathione at the cysteinylglycine bond may be of metabolic significance. The present and earlier findings lead to a tentative scheme (presented here) for the metabolism and translocation of glutathione, gamma-glutamyl amino acids, and related compounds.

Animals↗
PubMed1979

Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase.

This work relates to the hypothesis that one of the mechanisms that mediates amino acid translocation across cell membranes involves the action of membrane-bound gamma-glutamyl transpeptidase on intracellular glutathione and extracellular amino acids to form gamma-glutamyl amino acids. According to this idea, the latter are translocated into the cell where the gamma-glutamyl moiety is removed to yield free amino acids. Previous studies in this laboratory showed that intracellular glutathione is translocated out of many cells. We have now directly examined the transport of gamma-glutamyl amino acids into tissues in the mouse by use of the model substrate L-gamma-glutamyl-L-[14C]methionine sulfone. Of 11 tissues examined, only the kidney showed strong and preferential uptake of the substrate. A substantial amount of the administered L-gamma-glutamyl-L-[14C]methionine sulfone was found intact in the kidney; the total uptake of this compound was greater (by about 2-fold) than that of free L-methionine sulfone. Studies with a number of other gamma-glutamyl amino acids and gamma-glutamyl compounds indicate that the kidney has a relatively specific transport system for gamma-glutamyl amino acids. Small but significant amounts of gamma-glutamylmethionine sulfone were found in the liver and pancreas, suggesting that other tissues may also have this system. Transport of gamma-glutamylmethionine sulfone into the kidney was inhibited by inhibitors of glutathione synthesis and of gamma-glutamyl transpeptidase. The results suggest that both the transpeptidase and glutathione may be involved in transport of gamma-glutamyl amino acids.

Amino Acids↗
PubMed1979

Translocation of glutathione from lymphoid cells that have markedly different gamma-glutamyl transpeptidase activities.

Translocation of intracellular glutathione to the medium was studied in lymphoid cells (grown in tissue culture) that have very high, very low, or intermediate levels of membrane-bound gamma-glutamyl transpeptidase, in the absence and presence of various inhibitors of this enzyme. The data show that glutathione is translocated to the medium by all of the cell lines studied, but that glutathione does not accumulate in the medium unless the cellular transpeptidase activity is either very low or substantially inhibited. Translocation of glutathione does not seem to be directly related to the activity of gamma-glutamyl transpeptidase. The present and previous [Griffith, O.W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, 268--272] findings suggest that translocation of intracellular glutathione is a general property of many mammalian cells. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell in the form of transpeptidation or degradation products. Translocation of glutathione may also reflect operation of a rather general mechanism that protects and maintains the integrity of cell membranes.

Biological Transport↗
PubMed1978

Differential inhibition of glutamine and gamma-glutamylcysteine synthetases by alpha-alkyl analogs of methionine sulfoximine that induce convulsions.

The alpha-methyl and alpha-ethyl analogs of methionine sulfoximine, like methionine sulfoximine, induce convulsions in mice and inhibit glutamine synthetase irreversibly; alpha-ethylmethionine sulfoximine is approximately 50% as inhibitory as methionine sulfoximine and alpha-methylmethionine sulfoximine. However, whereas alpha-methylmethionine sulfoximine and methionine sulfoximine inhibit gamma-glutamylcysteine synthetase markedly, alpha-ethylmethionine sulfoximine does not, nor does administration of the alpha-ethyl analog produce the decrease in tissue glutathione levels found after giving methionine sulfoximine or its alpha-methyl analog. The findings strongly indicate that methionine sulfoximine-induced convulsions are closely associated with inhibition of glutamine synthetase rather than with inhibition of gamma-glutamylcysteine synthetase. The alpha-alkyl methionine sulfoximine analogs cannot be catabolized via the corresponding alpha-keto or alpha-imino acids, and, like other alpha-substituted amino acids, are probably not metabolized to a significant extent in vivo; this suggests that the amino acid sulfoximine molecules themselves, rather than their metabolites, are directly involved in the induction of convulsions. Possible explanations for the reported lack of correlation between the occurrence of convulsions and the levels of glutamine synthetase activity (and its substrates and product) are considered. The findings suggest that studies on the mechanism of induction of convulsions may be extended significantly and refined in biochemical terms by the use of other structurally modified convulsant molecules.

Amino Acids↗
PubMed1978

Evidence that the gamma-glutamyl cycle functions in vivo using intracellular glutathione: effects of amino acids and selective inhibition of enzymes.

The function of the gamma-glutamyl cycle was explored in in vivo studies in which amino acids and specific inhibitors of cycle enzymes (gamma-glutamyl transpeptidase, gamma-glutamyl cyclotransferase, gamma-glutamylcysteine synthetase, and 5-oxoprolinase) were administered to mice. The findings, which show that the gamma-glutamyl cycle functions in vivo, support the conclusion that gamma-glutamyl amino acids formed by gamma-glutamyl transpeptidase from externally supplied amino acids and intracellular glutathione are translocated into the cell and thus indicate that there is a significant physiological connection between the metabolism of glutathione and the transport of amino acids.

Amino Acids↗
PubMed1977

Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites.

Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.

Adenine Nucleotides↗
PubMed1977

Selective inhibition of gamma-glutamyl-cycle enzymes by substrate analogs.

Substrate analogs have been obtained that selectively inhibit the reactions of the gamma-glutamyl cycle or that are susceptible to only limited metabolism by the cycle. Thus, glutathione synthesis may be inhibited and analogs of glutathione may be synthesized that do not participate in transpeptidation. Specific inhibitors of gamma-glutamylcyclotransferase and 5-oxoprolinase have been obtained. The findings offer new approaches to the in vivo study of the cycle and also to the design of more specifically directed analogs of inhibitors such as methionine sulfoximine and 6-diazo-5-oxonorleucine.

Acyltransferases↗