To assess the extent to which low hepatic gamma-cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of gamma-cystathionase activity with propargylglycine on the metabolism of L-[35S]methionine was determined in studies with freshly isolated rat hepatocytes. gamma-Cystathionase activity was inhibited 25%, 42%, 63% and 76% (maximal inhibition) by treatment with 2.5 mumol/L, 0.01 mmol/L, 0.02 mmol/L and 2 mmol/l propargylglycine, respectively. Inhibition of gamma-cystathionase activity with up to 0.02 mmol/L propargylglycine had no statistically significant effect on [35S]glutathione, [35S]sulfate or [35S]cysteine formation from [35S]methionine. However, treatment of cells with 2 mmol/L propargylglycine markedly inhibited the metabolism of [35S]methionine to [35S]glutathione by 93%, to [35S]sulfate by 88% and to [35S]cysteine by 89%; [35S]cystathionine accumulation in these incubation systems was 60 times control. Hepatic gamma-cystathionase activity in premature infants has been reported to be about 23% of mature levels (Zlotkin and Anderson, 1982; Pediatr. Res. 16: 65-68); this level of gamma-cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway. No evidence for cysteine synthesis from serine and sulfide, which can be catalyzed by cystathionine beta-synthase, or for methionine metabolism by an S-adenosylmethionine-independent pathway was obtained.

The metabolism of cysteine and cysteinesulfinate was studied in freshly isolated hepatocytes from fed rats and cats. In incubations of rat hepatocytes with cysteinesulfinate, the rate of hypotaurine plus taurine production was approximately the same as the rate of conversion of the 1-carbon of cysteinesulfinate to CO2. In contrast, no significant production of hypotaurine plus taurine occurred in incubations of cat hepatocytes with cysteinesulfinate. These data are consistent with the species difference in the activity of hepatic cysteinesulfinate decarboxylase, which converts cysteinesulfinate to hypotaurine. In incubations of either rat or cat hepatocytes with cysteine, no hypotaurine plus taurine production was detected. However, the 1-carbon of cysteine was converted to CO2 and the production of urea plus ammonia nitrogen was significantly increased over the rates observed in incubations of cells without substrate. Our results suggest that most cysteine oxidation by hepatocytes occurs by pathways that do not involve formation of cysteinesulfinate.

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80O% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mm-or 25 mM-cysteinesulphinate. In similar incubations with 1 mm-or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14C02 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production ofammonia plus urea N; cysteinesulphinate catabolism was not affected. These data suggest that a substantial proportion of cysteine is catabolized by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.

The metabolism of L-cysteine was studied in freshly isolated rat hepatocytes. Because cysteine is rapidly oxidized in oxygenated incubation medium at neutral pH, the effect of bathocuproine disulfonate, a copper-specific chelator, was investigated. Addition of bathocuproine disulfonate resulted in a higher extracellular cysteine-to-half-cystine ratio in incubations of hepatocytes with cysteine. Bathocuproine disulfonate also increased the total uptake and metabolism of cysteine plus cystine [cyst(e)ine] by hepatocytes, which is consistent with the more efficient transport of cysteine than of cystine by freshly isolated rat hepatocytes. The partitioning of cysteine between cysteinesulfinate-dependent and cysteinesulfinate-independent pathways of catabolism was also altered by the addition of bathocuproine disulfonate; the percentage of total catabolic flux that resulted in taurine plus hypotaurine formation was greater, and the percentage of total catabolic flux that occurred by the beta-cleavage of cystine in a reaction catalyzed by gamma-cystathionase was less in incubations that contained bathocuproine disulfonate. Thus addition of bathocuproine disulfonate to maintain a higher extracellular thiol-to-disulfide ratio favored cysteinesulfinate-dependent catabolism of cysteine in rat hepatocytes.