Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis.
Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.
D000302:Adrenal Cortex / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System; D004586:Electrophoresis; D006418:Heme; D007425:Intracellular Membranes / Q000378:metabolism*; D008081:Liposomes; D008928:Mitochondria / Q000648:ultrastructure*; D010766:Phosphorylation; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D008081:Liposomes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D013252:Steroid 11-beta-Hydroxylase; D014357:Trypsin