The long range goal is to provide a detailed understanding of the biochemistry and regulation of hepatic cytochrome P-450 enzymes which catalyze oxidative metabolism of steroids and other lipophilic endogenous compounds, using the rat as a model system. The focus of the current project period is on two steroid hydroxylase cytochrome P-450 enzymes that are regulated in a manner distinct from other hepatic P-450s. These are P-450Ch.7a' which which catalyzes 7-alpha hydroxylation of cholesterol, the first and rate-limiting step in the degradation of cholesterol to bile acids, and P-450 2a, which catalyzes steroid hormone 6-beta hydroxylation leading to deactivation and elimination of testosterone, progesterone and related compounds. The major objectives are (1) to assess the respective contributions of reversible enzyme phosphorylation and changes in enzyme polypeptide levels to the modulation of P-450Ch7a activity in response to dietary and hormonal factors, (2) to characterize the phosphorylation of P-450 2a both in vitro and in vivo and then assess the possible importance of this step for modulation of P-450 2a activity, and (3) to evaluate the roles of endogenous corticosteroids and growth hormone secretory patterns in the age and sex-dependent expression of P-450 2a. These studies should provide a detailed understanding of the regulation of P-450Ch7a and its role in cholesterol homeostasis, and may thereby suggest useful biochemical approaches to the control of a number of pathological conditions, including atherosclerosis and cholesterol gallstones. These studies should also lead to a better understanding of the possible role of reversible phosphorylation as a mechanism for differential regulation of the multiple cytochrome P-450 enzymes expressed in rat hepatic tissue and, in addition, may provide further insight into the mechanisms of hormonal regulation of the corresponding human enzymes.
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