Biochemical characteristics of purified beef liver NADPH–cytochrome P450 reductase

AbstractNADPH–cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent‐solubilized microsomes on first and second DEAE‐cellulose columns, followed by 2′,5′‐ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2′‐AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239‐fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 ± 3000 (N = 5) by SDS‐PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis–Menten kinetics, and the apparent Km of the purified enzyme was found to be 47.7 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37°C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH–cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N‐demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:286–297, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10054