STRUCTURE AND REGULATION OF CYTOCHROMES P-450

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Investigation of steroidogenic cytochromes P-450 will be continued with further characterization of the active sties of the six steroidogenic cytochromes P-450 (aromatase, 11B- hydroxylase, C27 side-chain cleavage, 21-hydroxylase, 17a- hydroxylase and C21 side-chain cleavage). Characterization will be achieved by active site-affinity-labelling using bromoacetoxy- steroids that bind specifically to the active sites of these enzymes. The enzymes are presently being subjected to the combined approaches of microsequencing and genetic cloning; these procedures will be continued until the complete sequences of these porcine enzymes are determined; this information is vital to interpretation of active site labelling. The organization of steroidogenic proteins (P-450, P-450 reductase, cytochrome b5) within testicular and adrenal microsomes from pig and rat will be studied to characterize a pregnenolone-binding protein, to cross- link cytochromes P-450 to adjacent microsomal proteins and to determine the arrangement of these proteins by immunoelectron microscopy. These studies of microsomal proteins will make use of well characterized antibodies that cross-react with the above proteins from pig and rat microsomes.