Structural and functional differences among purified recombinant mammalian aromatases: glycosylation, N-terminal sequence and kinetic analysis of human, bovine and the porcine placental and gonadal isozymes.
Recombinant porcine gonadal and placental, and the human and bovine, isozymes of aromatase cytochrome P450 (P450arom) were over-expressed in insect cells, purified and quantified by difference spectroscopy. Human and bovine P450arom exhibited greater apparent molecular size than either porcine isozyme prompting an examination of N-linked glycosylation and amino-terminal peptide sequence. Comparisons of substrate affinities and turnover were also made. In contrast to human and bovine P450arom which are N-linked glycoproteins, neither isozyme of porcine P450arom is glycosylated, explaining in part their lower molecular size. Differences found in N-terminal peptide sequences were unlikely to influence apparent molecular size or enzyme function. Human and bovine P450arom had similar affinities and turnovers for androstenedione (approximately 200 nM, 3/min) and testosterone (approximately 350 nM, 2/min). The porcine isozymes had 10-fold higher affinities but correspondingly lower turnovers, particularly the gonadal P450arom. Overall, the catalytic efficiency (Vmax/Km) was similar for all but porcine gonadal P450arom which was much lower. These data emphasize the structural and functional variability of even the most conserved of proteins among diverse species wherein such differences have previously remained unexpected.
Jo Corbin
C
C
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Mapes
S M
SM
Lee
Young M
YM
Conley
Alan J
AJ
D000595:Amino Acid Sequence; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D005260:Female; D006031:Glycosylation; D006066:Gonads / Q000201:enzymology*; D006107:Granulosa Cells; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013552:Swine; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D013739:Testosterone; D000735:Androstenedione; D001141:Aromatase