Abstract
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.
Highlights
Cytochrome P450SCC (CYP11A1) is an integral mitochondrial enzyme that is located on the matrix face of the inner membrane of mitochondria
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
This paper is available on line at http://www.jbc.org increased CHL binding to P450SCC caused by the branched PCs by performing CHL binding experiments to determine the kinetic parameters Km and Vmax for the side chain cleavage reaction as well as by direct spectral titration of the effect of branched PCs on the CHL binding to P450SCC
Summary
We show by stationary (gel chromatography) and time-resolved (stopped flow kinetics) experiments that the P450SCC membrane incorporation efficiency is greatly enhanced in the presence of branched PCs with regard to both the rate and the total extent of membrane association. We demonstrate by measurements of the P450SCC activity in primary and secondary vesicles that the branched PCs are stimulators of or at least retain rapid P450SCC exchange between vesicles (in contrast to CL, which partially inhibits this exchange). It is known that vesicles prepared by detergent removal and sonication often give nonreproducible results because residual detergent and vesicle size heterogeneity have profound influence on the properties of P450SCC mentioned above [2,3,4]. We used throughout the study large unilamellar vesicles prepared by the extrusion technique (LUVET), thereby taking the additional advantage of the very slow CHL exchange in these vesicles [3] and a fairly homogeneous size distribution of vesicles prepared this way [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
