STRUCTURE AND REGULATION OF CYTOCHROMES P-450

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We propose to purify adrenocortical microsomal cytochromes P-450 (17Alpha-hydroxylase; 21-hydroxylase) from pig. The enzymes will be characterized as proteins (purity, MW, isoelectric points), as heme proteins (spectra, substrate-induced difference spectra, heme content) and as enzymes (kinetics, inhibitors). Attention will be paid to comapring adrenal 17Alpha-hydroxylase with pig testicular microsomal P-450, which catalyzes both 17Alapha-hydroxylation and C(17,20)-lyase, and which we have recently purified. We plan to purify porcine cytochrome P-450 11Beta-/18-hydroxylase from the zona glomerulosa and zona fasiculata separately because our previous studies suggest that theree are two different enzymes in the cortex. The cholesterol side-chain cleavage enzyme (previously purified from beef) will also be purified from pig adrenocortical mitochondria. These enzymes will be sequenced in turn using automated microsequencing and cloning (Co-Principal Investigators J.E. Shively and B. Wallis, respectively). At the same time, active sites of these enzymes will be labeled by radioactive affinity alkylation using bromoacetoxy derivatives of the natural steroid substrates, and the alkylated amino acids will be identified and localized in the primary structure by the sequence studies. Binding proteins for pregnenolone in microsomes and for cholesterol in the inner mitochondrial membrane have been identified in the present period of support. These two proteins will now be isolated and characterized. Limited proteolysis of the three microsomal cytochromes P-450 (testicular hydroxylase/lyase; adrenal 21-hydroxylase and adrenal 17Alpha-hydroxylase) will be used to isolate a reductase-binding domain on P-450 using the binding fragment of the P-450 flavoprotein reductase attached to sepharose beads. The purified reductase-binding domains of different P-450's will be compared and the location of these peptides in the primary structures of the relevant P-450's will be determined. Collaborative extensions of the proposal will involve detailed studies by NMR and crystallography.