SIDE-CHAIN CLEAVAGE OF CHOLESTEROL AND CHOLESTEROL SULFATE BY ENZYMES FROM BOVINE ADRENOCORTICAL MITOCHONDRIA

The requirements for the conversion of cholesterol into pregnenolone and of cholesterol sulfate into pregnenolone sulfate (called side-chain cleavage), by fractions from bovine adrenocortical mitochondria, have been examined and found to be the same for both substrates. The requirements are reduced triphosphopyridine nucleotide, a reduced triphosphopyridine nucleotide-cytochrome P-450 reductase, nonheme iron, cytochrome P-450, and molecular oxygen. It is likely that some additional factor(s) may be required. Optimal conditions for side-chain cleavage were obtained with both substrates and the rates of cleavage with the two substrates were compared using three solvents or suspending agents, namely, N,N-dimethylformamide, bovine serum albumin, and Tween-80. Values for apparent Km and Vmax with standard errors were calculated by least-squares fit. The apparent Km for cholesterol sulfate (18 μm) is less than that for cholesterol (40-50 μm). Anomalous results were observed when cholesterol sulfate was suspended in Tween-80 and when cholesterol was added in N,N-dimethylformamide, probably as the result of inadequate saturation of the enzyme. Values for Vmax, with both substrates were of the same general order (1.5-5.9 mμ- moles/min per mg of protein). The sum of the cleavage of the two substrates incubated separately, approximately equals the total cleavage of both substrates incubated together. These observations suggest that under optimal conditions the two substrates are cleaved at about the same rate and that cholesterol and cholesterol sulfate are cleaved by separate enzymes. © 1969, American Chemical Society. All rights reserved.