Biochemical and Molecular Roles of Nutrients

Cysteine Concentration Regulates Cysteine Metabolism to Glutathione, Sulfate and Taurine in Rat Hepatocytes1 MARTHA H. STIPANÃœK,2 RELICARDO M. COLOSO,3 ROLANDO A. G. GARCIA4 AND MARK F. BANKS Diuision of Nutritional


Cornell University,

[35S]cysteine or L-[35S]methionine, cysteine formed from methionine was incorporated into hepatic pro teins more readily than cysteine given as such, whereas cysteine seemed to be used preferentially for hepatic glutathione synthesis. In contrast, methionine was found to be a much better source of cysteine for glutathione synthesis than was cysteine provided as such in studies with rat hepatocytes in primary culture (2). However, methionine was only slightly better than cysteine as a source of cysteine for metallothionein synthesis. In studies with freshly isolated rat hepatocytes, methionine has seemed to be su perior (3) or inferior (4, 5) to cysteine in supporting glutathione synthesis. Previous studies (6) of methionine, cysteine and L2-oxo-thiazolidine-4-carboxylate (OTC)5 metabolism by isolated rat hepatocytes demonstrated an apparent differential partitioning of the sulfur of these sub strates (cysteine or cysteine precursor) to glutathione, taurine and sulfate. At equimolar exogenous substrate concentrations, the percentage of total sulfurmetabolite production accounted for by glutathione

ABSTRACT The effect of cysteine concentration and cysteine source [cysteine, methionine or 2-oxothiazolidine-4-carboxylate (OTC)] on the metabolism of [35S)cysteine was studied in isolated rat hepatocytes. Production of each of the major metabolites of cysteine (ghitathione, sulfate, taurine) increased as cysteine or methionine, but not OTC, concentration in the medium was increased. At equimolar exogenous substrate con centrations, cysteine availability to hepatocytes was greater from exogenous cysteine than from methionine, and that from methionine was greater than from OTC. The partitioning of cysteine, or the percentage of total metabolism resulting in production of each of the major metabolites, was markedly affected by cysteine concen tration or availability. Low cysteine availability favored its utilization for ghitathione; high cysteine availability favored its catabolism to sulfate and taurine. Under con ditions of low cysteine availability (incubations with 0.2 mmol/L OTC), ghitathione, sulfate and taurine pro duction accounted for 90, 10 and 1%, respectively, of total metabolism. Under conditions of high cysteine availability (incubations with 1 mmol/L cysteine + bathocuproine disulfonate), glutalbione, sulfate and taurine production accounted for 19, 47 and 34%, re spectively, of total metabolism. Cysteine supplied as such and cysteine formed intracellularly from methionine were similarly partitioned. These studies demonstrate that methionine is not a superior substrate to cysteine for hepatic ghitathione synthesis and that cysteine con centration (presumably intracelhilar cysteine concentra tion) has a major effect on the partitioning of cysteine sulfur to taurine in rat hepatocytes. J. Nutr. 122: 420-427, 1992.

'Based upon work supported by the Cooperative State Research Service, U.S. Department of Agriculture under Agreement No. GAM900895 and by New York State Hatch Project 399472. Dr. Coloso was supported by a Fulbright-Hays Mutual Educational Exchange Program Scholarship Grant, Institute of International Education, New York, NY. ^To whom correspondence and reprint requests should be ad


•cysteine •glutathlone •sulfate •taurine


dressed. ^Present address: Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, P.O. Box 256, Hoilo City, Philippines. 4Present address: Unidad de Neurociencias, Instituto Interna cional de Estudios Avanzados, Apartado 17606, Parque Central, Caracas 1015-A, Venezuela. Abbreviations used: OTC, L-2-oxo-thiazolidine-4-carboxylate¡ BCS, bathocuproine disulfonate; DNFB, dinitrofluorobenzene; SSA, sulfosalicylic acid.

Several investigators have attempted to assess the relative contributions of the sulfur atoms of cysteine and methionine for the formation of various meta bolic products. In a study (1) with intact rats fed L0022-3166/92

Ithaca, NY 14853

$3.00 ©1992 American Institute of Nutrition. Received 26 April 1991. Accepted 26 August 1991. 420

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formation was 60% for OTC and 48% for methionine compared with only 28% for cysteine. The differ ential utilization of these substrates for glutathione synthesis seemed to be inversely correlated with the total rate of sulfur-metabolite production, suggesting that it was an effect of different intracellular cysteine concentrations generated by the various substrates. To further explore the utilization of cysteine for formation of these metabolites, we conducted a series of studies. In the first, the relative ability of cysteine, methionine and OTC to support formation of glutathione, sulfate and taurine was determined. In the second study, the partitioning of the sulfur of 35S-labeled cysteine or methionine was determined under conditions in which the molar concentrations of both methionine and cysteine were held constant to ensure constant intracellular concentrations of substrates and intermediates. In the third study, the effect of cysteine concentration on the partitioning of cysteine sulfur to glutathione, sulfate and taurine was determined.

MATERIALS AND METHODS Materials. Sources of substrates, radiolabeled com pounds and most reagents were reported previously (6). Catalase (EC and Percoli were obtained from Sigma Chemical (St. Louis, MO). L-2-Oxo[35S]thiazolidine-4-carboxylate was prepared from L[35S]cysteine by a modification of the method of Boettcher and Meister (7) as described previously (6). L-[35S]Methionine, L-[35S]cysteine and OTC were pu rified before use as described previously (6, 8, 9). Hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats (Blue Spruce Farms, Altamont, NY) that weighed -250 g. For at least 1 wk prior to hepatocyte preparation, rats were housed in indi vidual stainless steel mesh cages in a room main tained at 20°Cand 60-70% humidity with light from 0530 to 1730 h. Rats were fed a nonpurified diet with ad libitum access (Prolab RMH 1000, Agway, Syra cuse, NY). The care and use of animals was approved by the Cornell University Institutional Animal Care and Use Committee and conformed to Public Health Service/National Institutes of Health and U.S. Department of Agriculture standards. Hepatocytes were isolated by the method of Berry and Friend (10) as modified by Krebs et al. (11). In the third study, the cell suspension was further purified by mixing hepatocytes suspended in Krebs-Henseleit buffer (pH 7.4) with an equal volume of 9:1 Percoli: 1Ox-concentrated Krebs-Henseleit salt solution (no bi carbonate). The mixture was centrifuged at 6000 x g for 1.5 min at room temperature, and the supernatant was discarded. The cells were washed once with Krebs-Henseleit buffer to remove the Percoli, pelleted Downloaded from by Washington University, Law School Library user on 09 April 2018


by centrifugation and resuspended in Krebs-Henseleit buffer. Maintenance of a cellular ATP concentration of >2.0 |imol/g throughout the total incubation with various substrates was used as the criterion for via bility,- data for cells with lower ATP levels were dis carded. Incubations. Incubations were conducted as described previously (6, 12). In incubations with cys teine (or cysteine plus methionine), substrate rou tinely was prepared with bathocuproine disulfonate (BCS) to yield a final concentration of 0.5 mmol/L BCS in the incubation mixture. Incubations were stopped immediately after ad dition of hepatocytes (zero-time) and at 20 and 40 min (Studies 1 and 2) or at 10 and 20 min (Study 3); zerotime incubations served as blanks, and values for zero-time blanks were always subtracted from the 10- or 20-min values. Subtraction of the 20-min value from the 40-min value or of the 10-min value from the 20-min value gave a production rate for the second half of the incubation period. The shorter incubation times were used in Study 3 to assure linearity of metabolite production in incubations with substrate concentrations of

Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes.

The effect of cysteine concentration and cysteine source [cysteine, methionine or 2-oxo-thiazolidine-4-carboxylate (OTC)] on the metabolism of [35S]cy...
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