Abstract
NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.
Highlights
Was purified to apparent homogeneity from microsomes of house flies, Musca domesticu L
The key to this purification procedure is the use of an affinity column composed of agarose-hexane-nicotinamide adenine dinucleotide phosphate that has a high affinity for the house fly NADPH
The data reported here indicate that use of this procedure produced the most highly purified preparations of house fly NADPH-cytochrome c reductase ever reported
Summary
Was purified to apparent homogeneity from microsomes of house flies, Musca domesticu L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The V,,,,, and K,, for cytochrome c were 42.3 pmol min-’ mg-’ and 12.7. Were 42.4 pmol min-’ mg-’ and 5.0 PM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-I. NADP+ and 2’-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 pM, respectively.
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