Branched phosphatidylcholines stimulate activity of cytochrome P450SCC (CYP11A1) in phospholipid vesicles by enhancing cholesterol binding, membrane incorporation, and protein exchange.
Phosphatidylcholines (PCs) with branched fatty acyl chains substituted in the two positions of the main chains (branched PCs) have been shown to be potent activators of the side chain cleavage activity of cytochrome P450SCC (CYP11A1) (Schwarz, D., Kisselev, P., Wessel, R., Jueptner, O., and Schmid, R. D. (1996) J. Biol. Chem. 271, 12840-12846). The present study reports on the effect of a series of branched PC on cholesterol binding, membrane integration, and protein exchange in large unilamellar vesicles prepared by an extrusion technique. Enzyme kinetics using vesicles as well as optical titration using a micelle system with the detergent Tween 20 demonstrate that activation is correlated with the fraction of P450SCC in the high spin form. The potency of branched PCs both to activate the enzyme and to induce spin state changes increases with increasing lengths of both the branched and main fatty acyl chains. We found that the extent as well as the rate of integration of P450SCC into vesicle membranes studied by gel chromatography and stopped flow kinetics were increased by branched PC. Finally, it is demonstrated by measurement of the enzymatic activity in primary and secondary vesicles that branched PCs are potent in retaining a very rapid exchange of P450SCC between vesicles, in contrast to cardiolipin, that partially inhibits this exchange process. The data suggest that different properties of P450SCC in membrane systems including cholesterol binding, membrane integration, and protein exchange are affected by branched PCs and probably by other phospholipids, too, and therefore must be considered in an explanation of the observed high stimulation of activity.
Kisselev
P
P
Institute of Clinical Pharmacology, Charite-Humboldt University of Berlin, Berlin/Max Delbrueck Centrum for Molecular Medicine, Berlin-Buch, Germany.
Wessel
R
R
Pisch
S
S
Bornscheuer
U
U
Schmid
R D
RD
Schwarz
D
D
D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding
D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme