Different isozymes of mouse 11 beta-hydroxylase produce mineralocorticoids and glucocorticoids.
We have isolated and characterized two isozymes of mouse steroid 11 beta-hydroxylase (11 beta-OHase), designated 11 beta-OHase and aldosterone synthase (AS). Physical mapping of overlapping cosmid and phage isolates defined two genes (designated Cyp11b-1 and Cyp11b-2 in the standard nomenclature for cytochrome P450 genes) that are oriented in the same direction and separated by approximately 8 kilobase pairs of DNA. The two genes are highly homologous in their coding regions, with 84% nucleotide identity and 86% predicted amino acid identity. In regions where the sequences of the rat 11 beta-OHase and AS genes diverged most widely, the mouse sequences also differed significantly, thereby identifying putative mouse 11 beta-OHase and AS genes. Both genes were mapped to chromosome 15 by analyzing restriction fragment length variations in a panel of DNA samples from an interspecific cross. To determine the functional properties of the 11 beta-OHase and AS proteins, we transfected COS-7 cells with plasmids that expressed the proteins encoded by the 11 beta-OHase and AS genes. When expressed in transfected COS-7 cells, the 11 beta-OHase protein converted deoxycorticosterone to corticosterone but did not produce aldosterone. Consistent with its postulated role in mineralocorticoid biosynthesis, the product of the AS gene efficiently synthesized aldosterone. We next studied the expression of these two isozymes in Y1 adrenocortical tumor cells and in the intact mouse adrenal gland. Although Y1 cells otherwise resemble zona fasciculata cells and express the 11 beta-OHase gene at high levels, transcripts encoded by the AS gene were detected at levels approximately 10-fold lower than the 11 beta-OHase transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)
Domalik
L J
LJ
Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Chaplin
D D
DD
Kirkman
M S
MS
Wu
R C
RC
Liu
W W
WW
Howard
T A
TA
Seldin
M F
MF
Parker
K L
KL
D000306:Adrenal Cortex Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000450:Aldosterone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line / Q000166:cytology / Q000378:metabolism; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005938:Glucocorticoids / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008901:Mineralocorticoids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010449:Peptide Mapping; D012150:Polymorphism, Restriction Fragment Length; D012689:Sequence Homology, Nucleic Acid; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000378:metabolism / Q000473:pathology
D005938:Glucocorticoids; D007527:Isoenzymes; D008901:Mineralocorticoids; D000450:Aldosterone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase