Major advances have been made for the isolation, purification and physical characterization of the family of hemeproteins called cytochromes P-450. These have been accompanied by a rapid growth of our knowledge of the genomic information of some cytochromes P-450. Concomitant advances in our understanding of the enzymology of these hemeproteins, as they participate in the myriad of reactions related to drug metabolism and the oxidative biotransformation of other xenobiotics, has regrettably lagged behind. It is proposed to continue studies directed to better evaluate the enzymatic properties of reactions catalyzed by cytochromes P-450 as these proteins reside within the protein mix of the microsomal membrane. Major emphasis will involve the use of automated hplc methods to measure the metabolism of substrates capable of undergoing multiple sites of hydroxylation, such as the steroids testosterone, androstenedione, etc. It is proposed that steroids can serve as sensitive monitors of the function of """"""""individual"""""""" types of P-450's. Using preparations from liver of various species of animals treated with a variety of different inducing agents (to alter the isozyme composition), it is proposed to evaluate the enzymology of unique types of P-450's as perturbed by the simultaneous function of other types of P-450. One area of emphasis will involve the use of specific inhibitors (monoclonal antibodies, azole derivatives such as ketoconazole and clotrimazole, competitive drug substrates, as well as agents to modify electron-flow), together with a variety of steroid substrate derivatives. Combined with spectrophotometric studies and computer modeling experiments, such metabolite identification studies will allow the assessment of differences in enzymatic function of comparable P-450's present in different species, containing similar (or different) epitopes. A significant effort will be made to assess the effect of the nutritional status, in particular the role of polyunsaturated fatty acids, on the interaction of unique P-450's. These studies will be extended to determine whether areas of """"""""P-450 metabolic zonation"""""""" can be identified in vitro by using induction regiments involving two or more inducing agents simultaneously, e.g. hypolipidemic agents and pregnenelone-16 - carbonitrile, and then seeking to separate unique populations of membranes enriched in specific types of P-450. Companion studies will measure the simultaneous incorporation into membranes in vitro two or more P-450's synthesized from enriched mRNA fractions.
