The use of 8-aminooctyl sepharose for the separation of some components of the hepatic microsomal electron transfer system.
Hepatic microsomes from phenobarbital-pretreated rats were treated with sodium cholate in the presence of glycerol and the solubilized proteins were absorbed 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], NADPH-cytochrome c reductase [EC 1.6.2.4], 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 election transfer enzymes.
D000818:Animals; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D003579:Cytochrome Reductases / Q000302:isolation & purification* / Q000378:metabolism; D003580:Cytochromes / Q000302:isolation & purification* / Q000378:metabolism; D004579:Electron Transport; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D051381:Rats; D012685:Sepharose; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D003580:Cytochromes; D012685:Sepharose; D003577:Cytochrome P-450 Enzyme System; D010089:Oxidoreductases, N-Demethylating; D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase