High density lipoprotein cholesterol as a mechanistic probe for the side chain cleavage reaction

Cholesterol side chain cleavage reaction catalyzed by purified cytochrome P-450scc was stimulated 4-5 fold when cholesterol in rat high density lipoprotein was used as a substrate as compared to the case where cholesterol plus 0.1% Emulgen 911 was used. In the case of the cholesterol-Emulgen system, the Vmax value of activity was not obtained even when a 20 times molar excess of adrenodoxin over the cytochrome was used. However, in the case of the lipoprotein, a 2-3 times molar excess of adrenodoxin over the cytochrome was enough to obtain the half value of Vmax. HPLC gel filtration experiments showed that the three enzymes were eluted from the column as a complex regardless of adding the lipoprotein as judged by the activity and the blotting analysis. However, the activity with the lipoprotein was detected significantly earlier than that in the absence. These and other lines of evidence suggest that the lipoprotein vesicles promote a complex formation among the three enzyme components and serve as a probe for emphasizing a significance of a cluster mechanism in the reaction.