ACTH-induced increases in rabbit adrenal immunoreactive P-450(17 alpha) and P-450(21)

Rabbit adrenal 17 alpha-hydroxylase activity has previously been shown to increase dramatically following ACTH stimulation. The present study was designed to determine whether the increase in enzyme activity could be correlated with an increase in P-450(17 alpha) protein measured by immunoblotting using an anti-porcine P-450(17 alpha) antibody. It was found that the total and specific contents of rabbit adrenal immunoreactive P-450(17 alpha) were increased 6- to 8-fold and 4-fold, respectively, after ACTH stimulation. The results were similar whether the detection system was 125I-labeled protein A or an alkaline phosphatase-conjugated second antibody. Corresponding increases in 17 alpha-hydroxylase activity were also observed but were slightly less than the increases in immunoreactive P-450(17 alpha), suggesting that not all of the protein was enzymatically active. Comparatively, immunoreactive P-450(21) was increased only 1.3-fold. Antibodies to porcine P-450(17 alpha) and bovine P-450(21) reacted monospecifically with the homologous rabbit and guinea pig proteins as judged by the detection of single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition studies showed that in an assay using 125 micrograms per ml of microsomal protein ACTH-stimulated rabbit adrenal 17 alpha-hydroxylase activity was inhibited 72% at a 100 mg per ml concentration of the anti-porcine P-450(17 alpha); however, 47% inhibition was observed at the same concentration of anti-bovine P-450(21). Pre-immune IgG had no effect. Molecular weight, Mr, determinations by SDS-PAGE showed both rabbit and guinea pig P-450(17 alpha) to be 52 kDa; rabbit P-450(21), 54 kDa; and guinea pig P-450(21), 49 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)