Glutathione (GSH) plays a vital defensive role and modulates critical cellular processes. One important factor that determines the rate of GSH synthesis is the activity of gamma-glutamylcysteine synthetase (GCS). GCS is made up of a heavy and a light subunit (HS and LS), the former exhibits all of the catalytic activity of the holoenzyme but the latter makes the enzyme function more efficiently. We showed that hepatic GCS-HS is regulated transcriptionally by hormones and plating density of cultured cells and post-translationally by protein phosphorylation. The two subunits are differentially regulated. While hormones and density had no influence on the steady state mRNA level of GCS-LS, treatment of cultured rat hepatocytes with diethyl maleate (DEM), buthionine sulfoximine (BSO) and tert-butyl hydroquinone (ThH) increased the mRNA level and transcription (DEM and BSO) of both subunits. Using 2/3 partial hepatectomy (PH) to examine changes in hepatic GSH during growth, we found an early increase in GCS-HS EA level which contributed to the elevation in hepatic GSH. Blocking the increase in GSH with BSO adversely affected the course of liver regeneration. The following aims are direct extensions of these observations which will: 1) examine the mechanism of regulation of GCS-HS gene transcription by hormones, density, DEM and BSO - define the genomic structure of rat GCS-HS, localize the cis-acting regulatory elements and transcriptional factors involved in mediating transcriptional changes as a result of these treatments both in cultured hepatocytes and relevant in vivo models. Compare information obtained from the rat to that of the human promoter; 2) determine the site(s) of phosphorylation on the GCS-HS critical for GCS activity - identify critical site(s) of phosphorylation by site-directed mutagenesis; 3) examine the mechanism of the increase in GCS-LS gene transcription by DEM and BSO and the functional significance of hepatic GCS-LS - study gene regulation of the light subunit after various treatments using the same approach as aim #1, compare relative turnover rates of the two subunits and study the consequence of blocking GCS-LS synthesis; 4) characterize changes in GSH homeostasis during liver regeneration and cell cycle - elucidate the mechanisms for the increase in GSH and GCS-HS and examine the effect of cysteine or GSH ester supplement after PH. Evaluate the effect of BSO on the kinetics of the liver cell cycle and mechanisms of its inhibitory effect on the cell cycle in cultured hepatocytes. These studies should improve our understanding of GCS, an enzyme critical in cellular defense and growth.
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