The hamster adrenal cytochrome P450C11 has equipotent 11beta-hydroxylase and 19-hydroxylase activities, but no aldosterone synthase activity.
We have isolated a hamster adrenal P45OC11 cDNA which shared 90 and 84% homology, respectively, with the nucleotide sequence and the amino acid sequence of the hamster adrenal P450aldo. Both P450C11 and P450aldo cDNA coding sequences were inserted in the plasmid pBluescript SK, transcribed and then translated using a rabbit reticulocyte system in the presence of [35S]methionine. The reaction products were immunoprecipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis. A single 35S-labeled protein band was detected for P450C11 and for P450aldo, respectively. P450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment of ribosomes to mRNA. These constructions were transfected in COS-1 cells. 24 h after transfection, the presence of P450C11 and P450aldo mRNAs was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [14C]corticosterone, [14C]19-OH-deoxycorticosterone and [14C]18-OH-deoxycorticosterone in ratios of 1:1.11:0.07, after 2 h of incubation; no [14C]aldosterone could be detected. Cells transfected with plasmids harboring the P450aldo cDNA transformed [14C]deoxycorticosterone to [14C]corticosterone, [14C]aldosterone, [14C]18-OH-corticosterone, [14C]18-OH-deoxycorticosterone, [14C]19-OH-deoxycorticosterone and [14C]11-dehydrocorticosterone 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 ultimate step of glucocorticoid formation and a separate P450 is involved in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 11beta and 19 in nearly equal ratio makes this animal an excellent model to study the mechanism of synthesis and inhibition of 19-OH-deoxycorticosterone, the precursor of 19-nor-deoxycorticosterone, a very potent mineralocorticoid involved in the development of essential hypertension.
Véronneau
S
S
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Bernard
H
H
Cloutier
M
M
Courtemanche
J
J
Ducharme
L
L
Lefebvre
A
A
Mason
J I
JI
LeHoux
J G
JG
D000311:Adrenal Glands / Q000737:chemistry / Q000201:enzymology*; D000450:Aldosterone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003345:Corticosterone / Q000378:metabolism; D006224:Cricetinae; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D000882:Haplorhini; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012156:Reticulocytes / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D000450:Aldosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C026961:prostaglandin omega hydroxylases; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D003345:Corticosterone