Methods

Protein Purification-Adrenodoxin reductase and adrenodoxin were purified from beef adrenal cortex mitochondria as described previously (11, 12), and respective concentrations were established using extinction coefficients of 10.9 m"' cm" at 450 nm (13) and 11 mM" cm" at 414 nm (14). Cytochrome P-450,, was purified as described previously (7) using cholate extraction of mitochondrial membranes, ammonium sulfate fractionation, and chromatography on hexyl agarose. The purified cytochrome was concentrated to 50 to 80 PM by ultrafitration, dialyzed into side chain cleavage buffer (20 mM Hepes, pH 7.2, lOO m~ NaCl, and 0.1 mM dithiothreitol), aliquoted into small vials, and either used immediately for experiments or frozen and stored in liquid nitrogen. Enzyme stored in this manner retained most of its activity (Vm-10 min" compared with a Vm of 2 to 3 min" using enzyme stored at-20" C). The concentration of cytochrome P-450,, was determined from the reduced CO minus reduced difference spectrum, using a difference extinction coefficient of 91 II1"'cm" for A4m minus A~ w (15). All optical spectra were recorded using a Varian 219 spectrophotometer. Preparation of Phospholipid Vesicles-Small unilamellar vesicles were prepared as described previously (7). Phospholipids in chloroform or ether (0.2 to 2 mg of total lipid) were mixed in a test tube (13 X 100 mm) with various amounts of cholesterol in ether or hydroxycholesterols dissolved in chloroform, and solvent was removed by blowing under a gentle stream of dry nitrogen. Assay buffer (0.5 ml) of the indicated pH was added to each tube, and tubes were sealed under Nz and sonicated for 10 min using a Beuhler Ultramet I11 bathtype sonicator. Cytochrome P-450,, was reconstituted into vesicle membranes as described previously (7, 8) by addition of cytochrome (0.1 to 1 PM final concentration) to vesicles, and preincubation for 10 min at 25" C or 5 min at 37" C prior to spectrophotometry or catalytic assays.

Assay of Pregnenolone Formation-Cholesterol or hydroxycholesterol side chain cleavage to yield pregnenolone was assayed basically as described previously (9). The assay mixture consisted of the indicated concentration of cytochrome P-45O,,-phospholipid vesicle preparation, 0.5 p~ adrenodoxin reductase, 10 pM adrenodoxin, 2 mM neutralized glucose 6-phosphate, and 2 units/ml of glucose-6-phosphate dehydrogenase, all in a volume of 170 to 200 pi. At zero time, NADPH was added to 50 PM final concentration, and 30-pl aliquots were taken at 0, 2, 4, 6, and 8 min and pipetted into 1 ml of hexane. Tightly capped vials of hexane were then mixed on a Vortex mixer for 15 s and could then be stored at-20" C prior to pregnenolone analysis. Radioimmunoassay of pregnenolone was accomplished as described previously (9, 16). Each pregnenolone analysis was done in triplicate or more to ensure accuracy, and standard errors were typically less than 5% of the determined value. As in earlier studies (8), it was necessary to correct for loss of heme during the reconstitution procedure by measurement of the carbon monoxide difference spectrum of cytochrome P-450 in the reconstituted phospholipid vesicle preparation.