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

Abstract

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) 612-6141. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J . Biochem. 152,. This paper describes the purification of the pyrazoleinducible 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 FM 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.It has been suggested that the Coh locus regulates the 7-hydroxylation of coumarin (Coh for coumarin hydroxylase). This is based on the fact that the liver microsomal coumarin 7-hydroxylase activity varies among inbred strains of mice and is differently regulated from other liver microsomal P-450-dependent reactions [l]. A high microsomal coumarin 7-hydroxylase activity has also been found in rabbits, guinea-pigs, hamsters, pigs and humans [2-41. As a structural gene product of this locus, a so-far unknown cytochrome P-450 isoenzyme, capable of catalyzing the 7-hydroxylation of coumarin, was suggested to exist [5]. In 1985 Kaipainen et al. [6] from our laboratory gave the first direct evidence for the existence of this protein by purifying a cytochrome P-450 isoenzyme from liver microsomes of phenobarbital-pretreated D2 mice with a high affinity and specificity for coumarin 7-hydroxylation.Recently we found [7] that microsomal coumarin 7-hydroxylase is strongly increased in mice by pyrazole, a compound better known as an inhibitor of alcohol dehydrogenase. This induction is interesting in at least three different respects. First, the effect of pyrazole is much stronger than that of phenobarbital, the only inducer so far known to induce coumarin 7-hydroxylase. Second, the induction was strong only in D2 mice and weak or absent in B6 or AKR mice. Third, the effect on microsomal P-450-catalyzed reactions was selective, unlike that of phenobarbital, since only coumarin 7-hydroxylation (and to a lesser extent 7-ethoxycoumarin 0-deethylase) was increased by the compound while several other P-450-Correspondence to M. A. Lang, Farmakologian Laitos ja Toksikologian Laitos, Kuopion Yliopisto, Postilokero 6, SF-7021 1 Kuopio, Finland dependent reactions, in addition to the total microsomal P-450 content, were either not affe...

Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes

“…As we had observed previously in the case of P450 3A, related forms, we noticed large variations in the inducibility, more particularly with rifampicin. In the cultures, we did not observe any induction with pyrazole, which has been reported to induce coumarin hydroxylase in the mouse [40]. In accordance with the in vivo induction o f baboon, we noticed that phenobarbital was also a potent inducer of the P450 2A related protein in vitro.…”

Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B1 metabolism in human liver.

“…Hepatic levels of members of the P450IIIA gene family are regulated by glucocorticoids and a wide variety offoreign compounds (32). Recent studies have also indicated that in the mouse P450IIA proteins are highly inducible by pyrazole and to a lesser degree by phenobarbital (33). It is interesting that P450IIA proteins catalyze coumarin hydroxylation (34,35) and that AFB1 also contains the coumarin structure.…”

Human and mouse liver coumarin 7‐hydroxylases do not metabolize warfarin in vitro.

“…Electrophoretically homogeneous P450 2a-5 was prepared from pyrazole-treated DBA/2N mouse livers, and NADPH-cytochrome P450 reductase was prepared from phenobarbitone-treated rat livers, as described by Juvonen et al (1988) Kaminsky et al, 1984) were added. The reaction was started with 2 mm NADPH.…”