SIMULTANEOUS ISOLATION OF NADPH-CYTOCHROME P-450 REDUCTASE AND CYTOCHROME-P-450 USING TENTACLE ION-EXCHANGE CHROMATOGRAPHY AND INTERSPECIESCOMPARISON OF THE REDUCTASE-ACTIVITY

Using the same initial Fractogel (tentacle) ion-exchange chromatograph y to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P -450 reductase (EC 1.6.2.4) was simultaneously isolated from solubiliz ed liver microsomes and purified on a DE-52 column to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 77 kD, and its specific activity was 25.4 mu mol . min(-1). mg protein(-1). Purified constitutive mouse liver NADPH -cytochrome P-450 reductase was successfully reconstituted in vitro wi th dilauroylphosphatidylcholine and constitutive purified mouse testos terone 2 alpha-hydroxylase (cytochrome P-450(2 alpha)) with an observe d activity of 13.8 nmol . min(-1). nmol P-450(-1). Although the partia lly purified reductase obtained from the Fractogel column was contamin ated by significant levels of two unidentified proteins, it was as equ ally effective in the reconstituted system as the DE-52-derived purifi ed reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-4 50 reductases were similarly effective in supporting the catalytic act ivity of rat cytochrome P-450 2B1, but the murine reductase was 50% mo re effective than the rat reductase in a reconstituted system containi ng mouse cytochrome P-450(2 alpha).