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Biomedical subjects

O W Griffith

Publications and source records attributed to O W Griffith.

At least 109 records · Page 6Linked to original sources
PubMed1983

Cysteinesulfinate metabolism. altered partitioning between transamination and decarboxylation following administration of beta-methyleneaspartate.

L-Cysteinesulfinate, a quantitatively important catabolite of L-cysteine, is a substrate of both cysteinesulfinate decarboxylase and glutamate-oxaloacetate transaminase. The former enzyme initiates a pathway leading to taurine; the latter enzyme forms beta-sulfinyl-pyruvate, which spontaneously decomposes to pyruvate and SO2. In the present studies, the in vivo partitioning of cysteinesulfinate between these two pathways was evaluated by administering to mice L-[1-14C]cysteinesulfinate, which is metabolized to 14CO2 by both pathways, or L-[3-14C]cysteinesulfinate, which is converted to 14CO2 only if taurine is not formed. Within 6 h, respiratory 14CO2 accounted for 90% of the [1-14C]cysteinesulfinate injected, whereas only 18% of administered [3-14C]cysteinesulfinate was recovered as 14CO2. When the data are corrected for differences in the formation of 14CO2 from [1-14C]- and [3-14C]pyruvate and for a small formation of 14CO2 from radiolabeled hypotaurine, it is concluded that approximately 85% of administered cysteinesulfinate is decarboxylated to hypotaurine, whereas approximately 15% is transaminated. Of the hypotaurine formed, approximately 90% is oxidized to taurine. beta-Methylene-DL-aspartate, an irreversible inhibitor of glutamate-oxal-oacetate transaminase (Cooper, A.J.L., Fitzpatrick, S. M., Kaufman, C., and Dowd, P. (1982) J. Am. Chem. Soc. 104, 332-334) was given to mice with the expectation that conversion of cysteinesulfinate to hypotaurine would be increased. Surprisingly, the extent of cysteinesulfinate transamination increased about 3-fold. Additional studies indicate that beta-methyleneaspartate is a potent, irreversible inhibitor of purified rat liver cysteinesulfinate decarboxylase and that inactivation of the decarboxylase predominates over inactivation of the transaminase in vivo. Highly purified cysteinesulfinate decarboxylase is also shown to decarboxylate L-aspartate to beta-alanine and, very slowly, glutamate to gamma-aminobutyrate. The enzyme is not active toward alpha-methylcysteinesulfinate or alpha-methylaspartate; alpha-methyl-DL-[1-14C]cysteinesulfinate is not metabolized by the mouse.

Animals↗
PubMed1982

Interaction of 5-oxo-L-prolinase with nucleoside triphosphates. Evidence suggesting substrate-dependent conformational change.

5-Oxoprolinase catalyzes the coupled hydrolysis of ATP and 5-oxoproline to yield glutamate, ADP, and Pi; the reaction may be partially or completely uncoupled by structural modification of either substrate. In the present work, we found slow 5-oxoproline-dependent changes in the rates of hydrolysis of ITP, GTP, and UTP. For example, in the absence of 5-oxoproline, the enzyme catalyzes the hydrolysis of UTP at a rapid and constant rate. Following addition of 5-oxo-L-proline, the rate of hydrolysis decreases slowly; after about 25 min, a much slower and constant rate of hydrolysis is attained. This change in rate is associated with a decrease in Vmax and an increase in the Km for UTP. In similar studies with ATP, both Vmax and Km increase over a much shorter time period (less than 10 s). The findings indicate that 5-oxoprolinase is a hysteretic enzyme, and are consistent with the hypothesis that in the normal catalytic reaction, the binding of both ATP and 5-oxo-proline to the enzyme induces a conformational change that brings the substrates into a juxtaposition that facilitates the reaction.

Amidohydrolases↗
PubMed1982

Glutathione metabolism in resting and phagocytizing peritoneal macrophages.

The steady state GSH content of cultured mouse resident peritoneal macrophages was 34 +/- 5 pmol/microgram of cell protein. Intracellular GSH content decreased concomitantly with zymosan ingestion. The half-life of GSH decreased from 1.9 h in resting cells to 0.58 h during phagocytosis as determined by inhibition of GSH synthesis with buthionine sulfoximine. The decrease in GSH half-life was directly related to the extent of particle uptake. In cytochalasin D-treated cells, attachment of zymosan to the macrophage plasma membrane in the absence of particle interiorization was sufficient to stimulate GSH turnover. Efflux was the major route of GSH loss in [35S]cystine-labeled macrophages, and was enhanced 3-fold by a zymosan challenge. GSH was lost intact since resident macrophages lack gamma-glutamyl transpeptidase (less than 1 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of protein . h). Macrophages obtained from mice challenged in vivo with Corynebacterium parvum maintained higher intracellular GSH levels (50 +/- 5 pmol/microgram of cell protein) than did resident cells. The half-life of GSH in buthionine sulfoximine-treated C. parvum-elicited macrophages was 3.8 +/- 0.2 h while resting and 1.3 +/- 0.2 h during phagocytosis. C. parvum-elicited macrophages, in contrast to resident cells, contained sufficient levels of gamma-glutamyl transpeptidase activity to hydrolyze 55 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of cell protein . h. These studies indicate that phagocytosis and cellular activation have profound effects on GSH metabolism in macrophages.

Animals↗
PubMed1982

Arachidonic acid metabolism in glutathione-deficient macrophages.

Mouse resident peritoneal macrophages were treated with the glutathione (GSH) synthesis inhibitor buthionine sulfoximine to deplete intracellular GSH. The arachidonic acid metabolites released by the GSH-depleted macrophages in response to a zymosan challenge were analyzed by HPLC. Buthionine sulfoximine treatment resulted in inhibition of both prostaglandin E2 and leukotriene C synthesis that was directly related to the degree of GSH depletion. Macrophages in which GSH levels were reduced to 3% of normal exhibited reductions to 4% and 1%, respectively, in PGE2 and LTC formation. The total quantity of cyclooxygenase metabolites secreted by GSH-deficient macrophages was identical to that of control cells as a result of increased synthesis of prostacyclin and, to a lesser extent, 12-L-hydroxy-5,8,10-heptadecatrienoic acid. Total lipoxygenase products were decreased, however; increased formation of hydroxyicosatetraenoic acids only partially compensated for the deficit in leukotriene C production. These findings extent our earlier observations on the inhibition of leukotriene C synthesis in GSH-depleted macrophages and confirm with intact cells the previously suggested role of GSH in prostaglandin E2 formation.

Arachidonic Acid↗
PubMed1981

The role of glutathione turnover in the apparent renal secretion of cystine.

Previous studies with cystinuric dogs and humans have demonstrated that the amount of cystine excreted in the urine is, in some cases, larger than the amount of cystine removed from the plasma by glomerular filtration. It was concluded that the kidney must secrete cystine into the renal tubule. The present studies indicate that renal glutathione turnover constitutes a mechanism of cystine secretion which may account for a large fraction of the cystine burden in the mouse renal tubule. Mice administered L-arginine or L-lysine, inhibitors of cystine transport, excrete large amounts of cystine in their urine (approximately 15 mumol of cystine/mg of creatine). If the mice are pretreated with buthionine sulfoximine, an inhibitor of glutathione biosynthesis, glutathione turnover is substantially decreased, and the arginine- or lysine-induced cystinuria is reduced by 43 to 55%. The plasma cystine concentration following arginine or lysine administration is reduced less than 15% by buthionine sulfoximine. These findings suggest the in vivo operation of a cycle in which glutathione, synthesized from cysteine intracellularly, is transported into the tubule and oxidized to glutathione disulfide. Subsequent breakdown of glutathione disulfide by gamma-glutamyl transpeptidase and dipeptidase releases cystine within the tubule. In the absence of cystine transport defects or inhibitors, cystine is reabsorbed and reduced intracellularly to cysteine.

Amino Acids↗
PubMed1981

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism.

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.

Adenosine Diphosphate↗
PubMed1981

Inhibition of glutathione synthesis as a chemotherapeutic strategy for trypanosomiasis.

With the expectation that trypanosomal glutathione (GSH) plays a major protective role against the endogenous oxidant stress that results form high intracellular levels of H2O2, we sought to deplete Trypanosoma brucei brucei of their GSH through inhibition of its biosynthesis. Administration of buthionine sulfoximine (BSO), a reversible inhibitor of gamma-glutamylcysteine synthetase, to parasitemic mice resulted in a progressive decrease in trypanosome GSH content, such that parasites isolated after 5 h or BSO treatment contained 50% of normal values. When BSO administration was continued for 18 h (intraperitoneal injection of 4 mmol/kg every 1.5 h), parasitemias temporarily cleared. When inhibitory plasma levels of BSO were maintained for about 27 h, two out of six infected mice were cured and the rest had significantly prolonged survival. These findings demonstrate the potential value of GSH depletion for the treatment of trypanosomiasis.

Animals↗
PubMed1981

Depletion of glutathione selectively inhibits synthesis of leukotriene C by macrophages.

We have examined the role of glutathione synthesis and intracellular glutathione content in the formation of leukotriene C (LTC) by mouse peritoneal macrophages. For this purpose, we utilized the drug buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Thirty minutes after the addition of BSO (200 microM) to macrophage cultures, when glutathione synthesis was inhibited approximately 80%, the cells responded to a zymosan challenge with a normal release of LTC. During this period, intracellular glutathione stores were not significantly depleted. Cells exposed to BSO for 2 hr or more exhibited marked decreases in glutathione levels and a progressive inhibition of LTC synthesis. After exposure to BSO for 16 hr, intracellular glutathione was undetectable, and no LTC was synthesized by the cells. Treatment of macrophages with BSO for 16 hr had no effect on cell viability, phagocytosis, total release of arachidonic acid, or prostaglandin synthesis. However, an increased synthesis of hydroxyicosatetraenoic acids in BSO-treated cells compensated for the diminished production of LTC. We conclude that BSO produces a specific, time-dependent inhibition of LTC synthesis as a result of intracellular glutathione depletion. This is consistent with a biosynthetic pathway for LTC in which glutathione is a direct precursor of this arachidonic acid metabolite.

Animals↗
PubMed1981

Formation of gamma-glutamycyst(e)ine in vivo is catalyzed by gamma-glutamyl transpeptidase.

These studies indicate that gamma-glutamylcyst(e)ine, found in the urine of a patient with gamma-glutamyl transpeptidase deficiency and also in the urine of experimental animals injected with glutathione or with inhibitors of gamma-glutamyl transpeptidase, is formed by the action of gamma-glutamyltranspeptidase. The evidence demonstrates that transpeptidation between glutathione and cystine occurs in vivo and also that this reaction constitutes a significant physiological function of the enzyme. The appearance of large amounts of gamma-glutamylcyst(e)ine in the urine seems to reflect an inhibitory effect of glutathione on the transport of gamma-glutamylcyst(e)ine into cells. The findings also indicate that conversion of glutathione to gamma-glutamylcysteine by hydrolytic cleavage of the COOH-terminal glycine moiety of glutathione (or analogous cleavage of glutathione disulfide) is not a quantitatively significant pathway. The results reported here show that gamma-glutamyl transpeptidase activity is not completely absent in a patient found to have a deficiency of this enzyme and that the activity of the enzyme is not abolished in experimental animals treated with potent gamma-glutamyl transpeptidase inhibitors.

Animals↗
PubMed1980

Interaction of L- and D-3-amino-1-chloro-2-pentanone with gamma-glutamylcysteine synthetase.

The optical isomers of 3-amino-1-chloro-2-pentanone, which are the alpha-chloroketone analogs of L- and D-alpha-aminobutyrate, were synthesized and found to be highly potent irreversible inactivators of gamma-glutamylcysteine synthetase. These chloroketones are 20 to 30 times more active than L-2-amino-4-oxo-5-chlorpentanoate. L- and D-Glutamate, in the presence of Mg2+ or Mn2+, protect the enzyme against inactivation. The enzyme is almost completely inhibited by cystamine under conditions in which 0.5 mol of this compound is bound/mol of enzyme. Treatment of the enzyme with cystamne, which produces inhibition that is reversible by dithiothreitol, prevents the interaction of the new chloroketones, L-2-amino-4-oxo-5-chloropentanoate and methionine sulfoximine with the enzyme. The findings suggest that a sulfhydryl group at the active site interacts with the chloroketones and with cystamine and that the chloroketone inhibitors and cystamine bind to the enzyme as glutamine analogs. The data also suggest that a gamma-glutamyl-S-enzyme intermediate may be formed in the reaction catalyzed by this enzyme.

Animals↗
PubMed1980

L-gamma-(Threo-beta-methyl)glutamyl-L-alpha-aminobutyrate, a selective substrate of alpha-glutamyl cyclotransferase.

L-gamma(Threo-beta-methyl)glutamyl-L-alpha-aminobutyrate was was prepared and found to be an excellent substrate of gamma-glutamyl cyclotransferase; in contrast to gamma-glutamyl-glutamine and other good substrates of cyclotransferase, the new substrate is not acted upon by gamma-glutamyl transpeptidase. gamma-Glutamyl cyclotransferase converts the new substrate to alpha-aminobutyrate and 3-methyl-5-oxoproline; the latter compound is not a substrate of 5-oxoprolinase. These properties of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-aminobutyrate facilitate its use in selectively determining cyclotransferase activity in biological materials that have transpeptidase activity. Thus, the new substrate was used here for the determination of the cyclotransferase activity of homogenates of various mouse tissues. The new substrate was also used to examine gamma-glutamyl cyclotransferase activity in vivo; thus, the rate of respiratory 14CO2 formation after administration of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate to mice provides a valid measure of cyclotransferase activity. beta-Aminoglutaryl-L-alpha-aminobutyrate is a competitive inhibitor of cyclotransferase (apparent Ki, 0.6 mM). Administration of beta-amino-glutaryl-L-alpha-aminobutyrate to mice out only decreased the level of 5-oxoproline in the kidney of control mice, but also of mice in which kidney 5-oxoproline levels were increased by administration of methionine. Administration of beta-aminoglutaryl-L-alpha-aminobutyrate to mice decreased the in vivo metabolism of L-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate as indicated by a marked decrease in the rate of respiratory 14CO2 formation. The findings indicate that gamma-glutamyl cyclo-transferase is a major in vivo catalyst for the formation of 5-oxoproline.

Acyltransferases↗