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

Abstract

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 K M of cholesterol could not be determined. The apparent K M value of NADPH is 6.25 × 10 −4 M. A pH optimum was found at pH 9.5 (Tris-buffer) and a temperature optimum at 40°C. The metal ions Sr 2+ and Ba 2+ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg 2+ and Ca 2+ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn 2+, Cd 2+, Cu 2+ and by 10 and 100 mM Fe 2+, Mn 2+, Co 2+ and Ni 2+. 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.