Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1.
A monoclonal antibody specific for cytochrome P-450 1 that extensively (greater than 95%) inhibits the hepatic 21-hydroxylation of progesterone was used in a two-site immunoradiometric assay to estimate the concentration of cytochrome P-450 1 in microsomes prepared from 24 individual, untreated New Zealand White rabbits. The progesterone 21-hydroxylase activities of these microsomes ranged from 0.2 to 5.8 nmol min-1 mg microsomal protein-1. Scatchard analysis revealed similar slopes and thus apparent affinities between the antibody and microsome samples that varied greater than 10-fold in 21-hydroxylase activity. The maximal extent of binding of the antibody to different microsomal preparations was greater for microsomes exhibiting high as compared to low 21-hydroxylase activity, suggesting that the level of binding reflects the microsomal content of P-450 1. Quantitation was based on the extent of binding of the 125I-labeled monoclonal antibody to P-450 1 sequestered from a sample by a heterologous monoclonal antibody adsorbed to the wells of a microtiter plate. These results indicate that the microsomal content of P-450 1 varies from less than 0.05 to 0.5 nmol/mg microsomal protein. The microsomal content of this antigen as determined in the two-site immunoradiometric assay was highly correlated (r = 0.97) with progesterone 21-hydroxylase activity. Linear regression analysis was used to estimate the turnover number for progesterone in situ, yielding a value of 11 nmol deoxycorticosterone formed min-1 nmol microsomal P-450 1(-1). This is similar to the value of 14 nmol deoxycorticosterone formed min-1 nmol-1 obtained for the reconstituted, purified P-450 1 used as a standard in the immunoquantitation assay.
D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007120:Immunochemistry; D008862:Microsomes, Liver / Q000201:enzymology*; D011817:Rabbits; D011863:Radioimmunoassay; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase