Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat liver microsomes

doi:10.1016/0003-9861(80)90119-8

Archives of Biochemistry and Biophysics

Volume 205, Issue 2, December 1980, Pages 365-379

https://doi.org/10.1016/0003-9861(80)90119-8 Get rights and content

Abstract

A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems.

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^☆: This research was supported in part by USPHS Grants ES 00267, ES 01590, and ES 02205. F.P.G. is the recipient of a USPHS Research Career Development Award (ES 00041).

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Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat liver microsomes☆

Abstract

Pharmacol. Ther. A

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Biol. Chem

Chem. Biol. Interact

J. Biol. Chem

Biochem. Pharmacol

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

Fed. Eur. Biol. Soc. Lett

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Pharmacol. Ther. A

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

Anal. Biochem