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J. Biochem, 1976, Vol. 80, No. 2 267-276
© 1976 Japanese Biochemical Society

research-article

The Use of 8-Aminooctyl Sepharose for the Separation of Some Components of the Hepatic Microsomal Electron Transfer System

Yoshio IMAI

Institute for Protein Research, Osaka University Suita, Osaka 565

Hepatic microsomes from phenobarbital-pretreated rats were treated with sodium cholate in the presence of glycerol and the solubilized proteins were adsorbed on a column of 8-aminooctyl derivative of Sepharose 4B. Washing the column stepwise with buffers containing suitable detergents in addition to cholate resulted in the elution of cytochrome P-450, NADH-cytochrome b5 reductase [EC 1.6.2.2 [EC] ], NADPHcytochrome c reductase [EC 1.6.2.4 [EC] ], and cytochrome b5; the former two enzymes were eluted with Emulgen 913 (a polyoxyethylene nonylphenyl ether), and then the latter two were obtained separately by increasing the concentration of added deoxycholate. Thus, cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome b5 were purified to specific contents (or activities) of 9–10 nmoles per mg of protein, 15–16 units per mg of protein, and nearly 35 nmoles per mg of protein, respectively (about 40–50% pure), in 40–50% yields. Practically no mutual contamination of the three enzymes was detected. Benzphetamine N-demethylation activity could be reconstituted on mixing the cytochrome P-450 and NADPH-cytochrome c reductase preparations in the presence of cholate. NADH-cytochrome c reductase activity could be observed at high efficiency under appropriate conditions in a system containing the cytochrome b5 and NADH-cytochrome b5 reductase preparations. Various derivatives of Sepharose 4B were compared for effectiveness in the separation of the microsomal electron transfer enzymes.


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