Characterization of human cholesterol side chain cleavage enzyme (EC 1.14.15x) of human term placental mitochondria.

The cholesterol side chain cleavage enzyme (EC 1.14.15x) in mitochondria of a human term placenta was partially characterized. Enzyme activity was determined by separation of [26-14C]-cholesterol and [5-14C]-isocaproic acid formed by side chain cleavage. Since the amounts of unlabeled cholesterol were too large, a KM of cholesterol could not be determined. The apparent KM value of NADPH is 6.25 x 10(-4) M. A pH optimum was found at pH 9.5 (Tris buffer) and a temperature optimum at 40 C. The metal ions Sr2+ and Ba2+ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg2+ and Ca2+ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn2+, Cd2+, Cu2+ and by 10 and 100 mM Fe2+, Mn2+, Co2+ and Ni2+. During preincubation of the enzyme without radioactive substrate, a rapid loss in enzyme activity in relation to enzyme concentration was observed (initial activity = 100%) (preincubation time in hours): 0.5 h (97%), 1 h (55%) and 1.5 h (34%). A dose-dependent inhibition of the enzyme by the following proteins was achieved: bovine serum protein, human serum protein, human immunoglobulin G and ovalbumin. Furthermore, a dose-dependent inhibition was found with the membrane lipids lecithin and sphingosine.