The structure and activity of two cytochrome P450c21 proteins encoded in the ovine adrenal cortex.
The steroidogenic enzyme cytochrome P450c21 (CYP21A1) is synthesized in the adrenal cortex and is essential for cortisol and aldosterone production. We have studied the structure and activity of ovine P450c21 proteins by analysis and expression of the corresponding cDNAs. Two P450c21 mRNAs (2.2 and 1.7 kilobases) were detected in ovine adrenal RNA and corresponded to two types of P450c21 cDNA clones that differed in their 3' region. One clone encoded a protein similar in structure to bovine, murine, and human P450c21 proteins. The other clone contained a 3' deletion of about 500 nucleotides and encoded a P450c21 protein that was truncated by 18 residues at the carboxyl terminus. The boundaries of this deletion suggested that an additional splicing event was responsible for the shortened mRNA sequence. Detailed Southern analysis of ovine genomic DNA indicates that the two mRNAs are derived from one gene even though two P450c21 genes are present in the ovine genome. The activities of the two P450c21 proteins were determined by expressing the respective cDNA clones in COS cells. The complete P450c21 protein was an efficient catalyst of 21-hydroxylation reactions, whereas no 21-hydroxylation activity was detected in cells containing the P450c21 protein with the carboxyl-terminal deletion.
Crawford
R J
RJ
Howard Florey Institute of Experimental Physiology and Medicine University of Melbourne, Parkville, Victoria, Australia.
Hammond
V E
VE
Connell
J M
JM
Coghlan
J P
JP
D000302:Adrenal Cortex / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D013255:Steroid 21-Hydroxylase