THE HAMSTER ADRENAL CYTOCHROME P450C11 HAS EQUIPOTENT 11-BETA-HYDROXYLASE AND 19-HYDROXYLASE ACTIVITIES, BUT NO ALDOSTERONE SYNTHASE ACTIVITY

We have isolated a hamster adrenal P450C11 cDNA which shared 90 and 84 % homology, respectively, with the nucleotide sequence and the amino a cid sequence of the hamster adrenal P450aldo. Both P450C11 and P450ald o cDNA coding sequences were inserted in the plasmid pBluescript SK, t ranscribed and then translated using a rabbit reticulocyte system in t he presence of [S-35]methionine. The reaction products were immunoprec ipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were a nalyzed by polyacrylamide gel electrophoresis. A single S-35-labeled p rotein band was detected for P450C11 and for P450aldo, respectively. P 450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment o f ribosomes to mRNA. These constructions were transfected in COS-1 cel ls. 24 h after transfection, the presence of P450C11 and P450aldo mRNA s was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [C-14]cort icosterone, [C-14]19-OH-deoxycorticosterone and [C-14]18-OH-deoxycorti costerone in ratios of 1:1.11:0.07, after 2 h of incubation; no [C-14] aldosterone could be detected. Cells transfected with plasmids harbori ng the P450aldo cDNA transformed [C-14]deoxycorticosterone to [C-14]co rticosterone, [C-14]aldosterone, [C-14]18-OH-corticosterone, [C-14]18- OH-deoxycorticosterone, [C-14]19-OH-deoxycorticosterone and [C-14]11-d ehydrocorticosterone in ratios of 1:0.25:0.45:0.04:0.04:0.04 after 12 h of incubation. These results indicate that one P450 catalyzes the ul timate step of glucocorticoid formation and a separate P450 is involve d in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 1 1 beta and 19 in nearly equal ratio makes this animal an excellent mod el to study the mechanism of synthesis and inhibition of 19-OH-deoxyco rticosterone, the precursor of 19-nor-deoxycorticosterone, a very pote nt mineralocorticoid involved in the development of essential hyperten sion. (C) 1996 Elsevier Science Ltd.