Abstract
Cholesterol side-chain cleavage (CSCC) activity towards exogenous cholesterol was quantified by an one-step reversed-phase minicolumn method for the separation of pregnenolone formed in the reaction. The assay is rapid and reproducible. The method is linear for up to 2 mg of placental mitochondrial protein and up to 1 mg of bovine adrenal mitochondrial protein in the incubate over 30 min and 5 min reaction times, respectively. Average Km and Vmax values were 14.1 μM and 3.4 pmol/min/mg for the placental preparation and 1.5 μM and 20.7 pmol/min/mg for the bovine adrenal mitochondrial preparation. In human placenta, the mitochondrial fraction contained most of the CSCC activity. Inhibition studies showed that aminoglutethimide (500 μM) inhibited both placental and bovine adrenal activities at the same level (about 80–90 % inhibition) but androstenedione (500 μM), metyrapone (500 μM), benzo(a)pyrene (800 μM) and Emulgen 911 (0.05 %) were more effective in human placental preparations. Neither of the activities were inhibited to any great extent by α-naphthoflavone (500 μM), SKF 525A (500 μM) or 7-ethoxycoumarin (1 mM).
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