Biochemical characterization of a truncated form of CYP27A purified from rabbit liver mitochondria.
During purification of CYP27A from rabbit liver mitochondria, a cytochrome P450 of different molecular size was co-isolated. The latter enzyme has an apparent M(r) 51,000 which is slightly lower than that of CYP27A. The 51,000-M(r) protein was found to be present in mitochondria from liver, small intestine, kidney, and spleen but not in lung, testis, heart, or brain mitochondria. Determination of the N-terminal sequence revealed that the 51,000-M(r) protein is a truncated form of CYP27A lacking the first 12 residues. The truncated enzyme was less efficient than the full-length CYP27A in the 27-hydroxylation of C(27)-sterols and much less efficient in the 25-hydroxylation of 1alpha-hydroxyvitamin D(3). The K(m) values for cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol were about the same with both enzymes whereas the K(m) for 1alpha-hydroxyvitamin D(3) was much higher with the truncated CYP27A. The results strongly indicate that the 51,000-M(r) protein is formed via proteolytic processing of CYP27A by endogenous protease(s) in some of the tissues examined. The truncation at the N terminus markedly impairs the ability of CYP27A to use 1alpha-hydroxyvitamin D(3) as substrate and to catalyze 25-hydroxylation in the bioactivation of vitamin D(3).
Furster
C
C
Department of Pharmaceutical Biosciences, University of Uppsala, Uppsala, S-751 23, Sweden.
Bergman
T
T
Wikvall
K
K
D000595:Amino Acid Sequence; D000818:Animals; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D013261:Sterols; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase