Cystathionase activity and glutathione metabolism in redifferentiating rat hepatocyte primary cultures

Capacity to incorporate methionine sulfur into glutathione as well as cystathionase activity were lost in cultured hepatocytes in a biphasic manner with 75% of the total capacity disappearing with a half-life of about 10.6 hr, the remainder with a half-life of greater than 20 hr. Nicotinamide, 25 mM, produced a single phase loss with a t 1/2 of approximately 21 hr for both transsulfuration and cystathionase activity. Loss of both methionine sulfur incorporation and cystathionase activity occurred in transferrin/sodium selenite-supplemented Williams Medium E (TS-HWME) with a t 1/2 of about 96 hr through 72 hr in culture. Addition of the cystathionase inhibitor, propargylglycine, blocked glutathione synthesis in TS-HWME cells through 48 hr in culture, while propargylglycine blocked glutathione synthesis only at 4 hr in HWME cultured cells. Further, the accumulation of gamma-glutamyl transpeptidase was delayed by 48 hr in TS-HWME versus unsupplemented medium. Variation in the transport of sulfur amino acids was also found to occur with culture age. The Km values for cysteine and methionine transport were found to be approximately 150 and 100 microM, respectively, and were unaffected by culture age or the presence of TS-HWME. However, the Vmax for transport of methionine declined from 0.29 to 0.012 nmol/min/mg protein over 48 hr in culture. In TS medium, the Vmax at 48 hr for methionine transport had only decreased to 0.20 nmol/min/mg protein and increased for cysteine transport to 0.17 nmol/min/mg protein. These data suggest that during the redifferentiation of hepatocytes in culture, transsulfuration is regulated by control of the flow of substrate through cystathionase and that cystathionase is regulated by alteration of enzyme activity or content. Variations in the rate of transport of precursor sulfur amino acids are also an important component of the regulation of the net glutathione status of the redifferentiating hepatocyte.