RECONSTRUCTION OF MITOCHONDRIAL CYTOCHROME P450-DEPENDENT STEROID HYDROXYLASES IN ARTIFICIAL PHOSPHOLIPID-MEMBRANES - STUDIES OF CYTOCHROMEP450SCC TOPOLOGY BY LIMITED PROTEOLYSIS

Highly purified cytochrome P450scc from bovine adrenal cortex mitochon dria was inserted in artificial phospholipid membranes prepared from p hosphatidylcholine to study the main principles of its membrane organi zation in the model system. Topology of the cytochrome P450scc polypep tide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of t he products of proteolysis products of the membrane-bound heme protein . It is shown that limited proteolysis of cytochrome P450scc in proteo liposomes results in a significant decrease of V-max for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted syst em in the presence of exogenously added adrenodoxin-reductase and adre nodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adr enodoxin is increased. Different models of membrane organization as we ll as functional specificity of cytochrome P450scc in artificial membr anes are discussed.