Purification and characterization of a liver microsomal cytochrome P-450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole-treated D2 mice.
Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-614]. Recently we found that in D2 mice this activity is strongly and selectivity induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152-3-8]. This paper describes the purification of the pyrazole-inducible cytochrome P-450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 microM coumarin in the preparations throughout the whole purification. Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.
Juvonen
R O
RO
Department of Pharmacology and Toxicology, University of Kuopio, Finland.
Shkumatov
V M
VM
Lang
M A
MA
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002845:Chromatography; D003374:Coumarins / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D013057:Spectrum Analysis; D013379:Substrate Specificity
D003374:Coumarins; D007527:Isoenzymes; D010743:Phospholipids; D011720:Pyrazoles; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6