Utilization of cysteine and cysteine precursors for the synthesis of glutathione in astroglial cultures: Preference for cystine
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Abstract:
The ability of astroglia-rich primary cultures derived from the brains of neonatal rats to take up and metabolize various sulfur containing compounds to cysteine was investigated using the content of intracellular glutathione as an indicator. Astroglial cells were partially depleted of glutathione by starvation for 24 h. Subsequent feeding for 4 h with glucose, glycine, and glutamate resulted in a restoration of the glutathione level, if cysteine was present. Substitution of cysteine by cystine during resynthesis of glutathione led to a glutathione content which exceeded that of cysteine-refed cells by 41%. Half-maximal content of glutathione was found at a concentration of about 12 microM cysteine and a maximal content at a concentration of at least 50 microM cysteine. In contrast, no plateau in the glutathione level was reached with increasing concentrations of cystine. The cystine effect could not be due to a contamination, since it was abolished after reduction of cystine by dithiothreitol. Since the cystine effect was not affected by inhibiting gamma-glutamyl transpeptidase, a promotion of cystine uptake by formation of gamma-glutamylcystine can also be excluded. Of the potential cysteine precursors tested, N-acetylcysteine was able to replace cysteine half-maximally at a concentration of 1 mM and fully at 5 mM. Feeding 2-oxothiazolidine-4-carboxylic acid at a concentration of 5 mM resulted in 64% of the glutathione level found in the presence of cysteine. A half-maximal glutathione content was attained at 50 microM 2-oxothiazolidine-4-carboxylic acid. While cystathionine could partially replace cysteine, methionine and homocysteine were not at all able to substitute for cysteine. These results demonstrate that astroglial cells prefer cystine from cysteine for glutathione synthesis and express uptake systems for N-acetylcysteine, 2-oxothiazolidine-4-carboxylic acid, and cystathionine, as well as the enzymes N-deacetylase, 5-oxoprolinase, and cystathionine gamma-lyase.Keywords:
Dithiothreitol
A study was conducted to determine free and protein-bound pools of glutathione and cysteine in the plasma of male broiler chicks. Birds were brooded in battery cages and provided ad libitum access to a starter diet and water. Plasma was treated with a reducing agent, dithiothreitol (DTT), or left untreated, and analyzed by HPLC to determine free and protein bound pools of reduced (GSH) and oxidized (GSSG) glutathione, cysteine (Cys), and cystine (Cyss). With respect to total plasma pools of GSH and Cys, between 0 and 21 d of age; 1) free GSH increased from 30 to 90% with a reciprocal decrease in protein-bound GSH, but GSSG was not detected; and 2) free Cys decreased from 20 to 10%, free Cyss increased from 24 to 45%, and protein-bound Cys decreased from 55 to 44%. The majority of the GSH plasma pool in this study was present in a free, acid-soluble form, whereas most of the total Cys pool was present as Cyss or bound to protein.
Dithiothreitol
Thiol
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Glutathione disulfide
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Bromobenzene
Cysteine Metabolism
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SummaryYoung male chicks were fed crystalline l-amino acid diets in assays designed to evaluate the capacity of d-and l-homocysteine to supply methionine and cysteine. l-Homocysteine had a cystine-sparing value of 100% and a methio-nine-sparing value of 28% at the level fed. d-Homocysteine was utilized less efficiently than was l-homocysteine, having a cystine-sparing value of 68% and a methionine-sparing value of only 7%.The methionine activity of l-homocysteine varied when evaluated in a cystine-adequate diet, being more efficiently utilized at low levels of intake. However, at every level fed, l-homocysteine was inferior to l-methionine in supporting growth. The presence of additional choline in the diet did not improve the performance of chicks fed l-homocysteine as a source of methionine. Performance of chicks fed dl-homocysteine as a source of total sulfur amino acids was equal to that of chicks fed dl-homocysteine as a source of methionine per se. The utilization of the d and l isomers of homocysteine appears to be limited by the methionine activity they can replace.The results of these assays clearly indicate that young chicks fed a crystalline l-amino acid diet cannot efficiently convert either d- or l-homocysteine to methionine via transmethylation.
Plasma homocysteine
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The glutathione content of freshly isolated rabbit alveolar type II cells, Clara cells and macrophages was depleted by incubating with diethyl maleate and the ability of these cell types to resynthesize glutathione from the sulfur-containing amino acids, cysteine, cystine and methionine was determined. Cysteine and methionine were taken up at a similar rate into each of the cell types, with cystine uptake occurring at a 10- to 14-fold lower rate. The cells were best able to utilize cysteine for glutathione resynthesis and the rates of both uptake and synthesis were fastest in macrophages and slowest in type II cells. There was no evidence for participation of a cystathionine pathway for glutathione synthesis in any of the cell types.
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SUMMARY Merino sheep were given abomasal infusions of various amino acids or mixtures of amino acids. Effects on wool growth were measured using autoradiography or a clipping procedure and changes in the concentration of amino acids in plasma were measured in some experiments. Mixtures of five (28 g/day) or ten (45 g/day) essential amino acids (both mixtures containing 3 g methionine) stimulated wool growth of sheep receiving a maintenance ration; on average, the volume of wool grown increased 48% and 86%, respectively. When cysteine completely replaced methionine in these mixtures, wool growth was markedly reduced, but two-thirds of the methionine could be replaced by cysteine without affecting wool growth. Homocysteine was partially effective in replacing methionine and, when supplemented with betaine, folic acid and vitamin B 12 , the mixture was still significantly inferior to that containing methionine. In contrast, abomasal supplements of methionine or homocysteine alone were equivalent as supplements for wool growth. The results indicated a specific role for methionine in the control of wool growth, other than the provision of cysteine. This role was postulated to be related to some function of S-adenosylmethionine. Infusion often essential amino acids caused appreciable increases in the concentrations of cystine, methionine, cystathionine and taurine in plasma; total essential amino acids increased threefold whereas nonessential amino acids decreased in concentration. The replacement of methionine in the infusion by cysteine or homocysteine significantly altered the concentration of cystine, methionine and cystathionine in plasma. Evidence was obtained that the adverse effects on wool growth of high abomasal doses of methionine (10g/day) could not be reduced or prevented by provision of additional glycine and were not related to the supposed toxic effects of 3-methylthiopropionic acid, a metabolite of the transamination pathway.
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Cysteine Metabolism
Glutathione disulfide
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Abstract Isolated hepatocytes lost about half their content of GSH when treated in vitro with 90 nmol of diethylmaleate per 10 6 cells. Within one hour GSH started to accumulate in the cells, and the rate of accumulation was taken as a measure of the GSH synthesis rate. The rate was affected by additions of amino acids and horse serum to the incubation medium. The methionine and cysteine uptake rates were much lower than the rate of GSH synthesis and not affected by variations in the GSH synthesis rate. The methionine and cysteine uptake rates were not affected by horse serum. It is concluded that even though exogenous sulphur‐containing amino acids facilitate GSH synthesis, the hepatic cysteine pool is to a large extent replenished by endogenous amino acids derived from protein degradation. In particular, this is the case when the turnover rate of the cysteine pool is increased by drug metabolism.
Cysteine Metabolism
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Glutathione disulfide
Cysteine Metabolism
Efflux
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Transsulfuration
Hyperhomocysteinemia
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