Phosphate → NADPH

Participants

Reaction Steps

Cell membrane fractions derived from insect cells transfected with recombinant baculoviruses expressing 11-hydroxylase and complementary electron transport proteins were analyzed for 11-hydroxylase activity in a reaction mixture containing 80 mM phosphate buffer, pH 7.4, 8 mM MgCl2, and 0.9 mM NADP+ in a final volume of 200 ul. In order to insure an adequate source of reducing equivalents, an NADPH regenerating system was provided by adding glucose-6-phosphate dehydrogenase (1.5 U/ml) and 8 mM glucose-6-phosphate. Steroid substrate (e.g., androstenedione) was provided at a final concentration of 0.3 mM. Reaction mixtures were incubated at 37° C. for 30 min. The reactions were terminated by the addition of 200 ul methanol and then placed on ice. Samples were pelleted by centrifugation to remove precipitated protein.

Provenance & Evidence