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The importance of cytochrome P-450 and P-448 in determining the specificity of the reconstituted liver microsomal hydroxylation system
287
Cher~-Bio£ Interactions
ElsevierPublishingCompany, Amsterdam Printed in The Netherlands
The importance of c y t o c h r o m e P-450 and P-448 in determining the specificity of the reconstituted liver microsomal hydroxylation system
R. KUNTZMAN,A.Y.H. LU, S. WEST,M. JACOBSONand A.H. CONNEY Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, N.J. 07110 (~s.a.)
Studies in several laboratories 1-4 have indicated that the microsomal CO-binding hemoprotein present in liver microsomes from 3-methylcholanthxene-treated rats is spectrally distinct from that found in untreated or phenobarbital-treated animals. Our findings, which showed that the Km for 3,4-benzpyrene hydroxylation was decreased by 3-methylcholanthrene treatment, prompted us to suggest that the spectrally distinct hemoprotein found in 3-methylcholanthrene-treated rats possessed an altered catalytic activitys . Evidence in support of this view has now been obtained in studies using the reconstituted microsomal hydroxylation system described by Lu and Coon6 . The liver microsomal hydroxylation system from phenobarbital- or 3-methylcholanthrene-treated male immature rats was solubilized and resolved into three fractions containing: (1) cytochxome I)-450 or P-448, (2) a NADPH-dependent reductase, and (3) lipid. All 3 fractions were required for maximum N-demethylation of benzphetamine or chlorcyclizine or for the hydroxylation of 3,4-benzpyrene or testosterone. The reconstituted system from phenobarbital-treated animals exhibited high activity for benzphetamine, chlorcyclizine and testosterone metabolism but very low activity for 3,4-benzpyrene hydroxylation. However, when the cytochxome I)-450 fraction from phenobarbital-treated rats was replaced by the cytochrome P-448 fraction from 3-methylcholanthxene-treated animals, a 20-fold increase in benzpyrene hydroxylase activity was observed, while the N-demethylation of benzphetamine and the 16-w hydroxylation of testosterone were markedly decreased. Moreover, the substitution of the cytochxome I)-448 fraction for the cytochxome P-450 fraction resulted in only a small decrease in the N-demethylation of chlorcyclizine and no significant change in the 7-w hydroxylation of testosterone. In contrast, the reconstituted system from 3-methylcholanthxene-treated rats showed good activity for the N-demethylation of chlorcyclizine and for both 3,4-benzpyrene hydroxylation and testosterone 7-a-hydroxylation but very low activity for the N-demethylation of benzphetamine and for testosterone 16-~-hydroxylation. Only the hydroxylation of 3,4-benzpyrene was markedly decreased by replacing the cytochrome P-448 fraction with a cytochrome P-450 fraction. These data suggest that the cytochrome P-450 and P-448 fractions have different catalytic activities. Studies to determine the role, if any, of the reductase fraction obtained from phenobarbital- or 3-methylcholanthrene-treated rats in determining substrate specifici.ty are now in progress.
Chem.-BioL Interactions, 3 (1971) 287-288
288
R. KUNTZMANet al.
REFERENCES 1 N.E. Sladek and G.J. Mannering, Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene-treated rats, Biochem. Biophy~ Re~ Commun., 24 (1966) 668. 2 A.P. Alvares, G. Schilling, W. Levin and R. Kuntzman, Alteration of the microsomal hemoprotein by 3-methylcholanthrene: effects of ethionine and actinomycin D, 3. PhannacoL ExptL Therap., 163 (1968) 417. 3 A. Hildebrandt, H. Remmer and R.W. Estabrook, Cytochrome P-450 of liver microsomes - one pigment or many, Biochem. Biophys. Re~ Cornmun., 30 (1968) 607. 4 Y. Gnosspelius, H. Thor and S. Orrenius, A comparative study of the effects of phenobarbital and 3,4-benzpyrene on the hyoroxylating system of rat-liver mierosomes, Chem.-BioL Interactions, 1 (1969/70) 125. 5 A.P. Alvares, G.R. Schilling and R. Kuntzman, Differences in the kinetics of benzpyrene hydroxylation by hepatic drug-metabolizing enzymes from phenobarbital- and 3-methylcholanthrene-treated rats, Biocher~ Biophyz Re~ Comraun., 30 (1968) 588. 6 A.Y.H. La and M.J. Coon, Role of hemoprotein P-450 in fatty acid O~-hydroxylationin a soluble enzyme system from liver microsomes, J. BioL Chem., 243 (1968) 1331.
Chem..BioL Interactions, 3 (1971) 287-288
Cher~-Bio£ Interactions
ElsevierPublishingCompany, Amsterdam Printed in The Netherlands
The importance of c y t o c h r o m e P-450 and P-448 in determining the specificity of the reconstituted liver microsomal hydroxylation system
R. KUNTZMAN,A.Y.H. LU, S. WEST,M. JACOBSONand A.H. CONNEY Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, N.J. 07110 (~s.a.)
Studies in several laboratories 1-4 have indicated that the microsomal CO-binding hemoprotein present in liver microsomes from 3-methylcholanthxene-treated rats is spectrally distinct from that found in untreated or phenobarbital-treated animals. Our findings, which showed that the Km for 3,4-benzpyrene hydroxylation was decreased by 3-methylcholanthrene treatment, prompted us to suggest that the spectrally distinct hemoprotein found in 3-methylcholanthrene-treated rats possessed an altered catalytic activitys . Evidence in support of this view has now been obtained in studies using the reconstituted microsomal hydroxylation system described by Lu and Coon6 . The liver microsomal hydroxylation system from phenobarbital- or 3-methylcholanthrene-treated male immature rats was solubilized and resolved into three fractions containing: (1) cytochxome I)-450 or P-448, (2) a NADPH-dependent reductase, and (3) lipid. All 3 fractions were required for maximum N-demethylation of benzphetamine or chlorcyclizine or for the hydroxylation of 3,4-benzpyrene or testosterone. The reconstituted system from phenobarbital-treated animals exhibited high activity for benzphetamine, chlorcyclizine and testosterone metabolism but very low activity for 3,4-benzpyrene hydroxylation. However, when the cytochxome I)-450 fraction from phenobarbital-treated rats was replaced by the cytochrome P-448 fraction from 3-methylcholanthxene-treated animals, a 20-fold increase in benzpyrene hydroxylase activity was observed, while the N-demethylation of benzphetamine and the 16-w hydroxylation of testosterone were markedly decreased. Moreover, the substitution of the cytochxome I)-448 fraction for the cytochxome P-450 fraction resulted in only a small decrease in the N-demethylation of chlorcyclizine and no significant change in the 7-w hydroxylation of testosterone. In contrast, the reconstituted system from 3-methylcholanthxene-treated rats showed good activity for the N-demethylation of chlorcyclizine and for both 3,4-benzpyrene hydroxylation and testosterone 7-a-hydroxylation but very low activity for the N-demethylation of benzphetamine and for testosterone 16-~-hydroxylation. Only the hydroxylation of 3,4-benzpyrene was markedly decreased by replacing the cytochrome P-448 fraction with a cytochrome P-450 fraction. These data suggest that the cytochrome P-450 and P-448 fractions have different catalytic activities. Studies to determine the role, if any, of the reductase fraction obtained from phenobarbital- or 3-methylcholanthrene-treated rats in determining substrate specifici.ty are now in progress.
Chem.-BioL Interactions, 3 (1971) 287-288
288
R. KUNTZMANet al.
REFERENCES 1 N.E. Sladek and G.J. Mannering, Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene-treated rats, Biochem. Biophy~ Re~ Commun., 24 (1966) 668. 2 A.P. Alvares, G. Schilling, W. Levin and R. Kuntzman, Alteration of the microsomal hemoprotein by 3-methylcholanthrene: effects of ethionine and actinomycin D, 3. PhannacoL ExptL Therap., 163 (1968) 417. 3 A. Hildebrandt, H. Remmer and R.W. Estabrook, Cytochrome P-450 of liver microsomes - one pigment or many, Biochem. Biophys. Re~ Cornmun., 30 (1968) 607. 4 Y. Gnosspelius, H. Thor and S. Orrenius, A comparative study of the effects of phenobarbital and 3,4-benzpyrene on the hyoroxylating system of rat-liver mierosomes, Chem.-BioL Interactions, 1 (1969/70) 125. 5 A.P. Alvares, G.R. Schilling and R. Kuntzman, Differences in the kinetics of benzpyrene hydroxylation by hepatic drug-metabolizing enzymes from phenobarbital- and 3-methylcholanthrene-treated rats, Biocher~ Biophyz Re~ Comraun., 30 (1968) 588. 6 A.Y.H. La and M.J. Coon, Role of hemoprotein P-450 in fatty acid O~-hydroxylationin a soluble enzyme system from liver microsomes, J. BioL Chem., 243 (1968) 1331.
Chem..BioL Interactions, 3 (1971) 287-288