Simulataneous isolation of NADPH-cytochrome P-450 reductase and cytochrome P-450 using tentacle ion-exchange chromatography and interspecies comparison of the reductase activity.
Using the same initial Fractogel (tentacle) ion-exchange chromatography to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was simultaneously isolated from solubilized 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 mumol.min-1.mg protein-1. Purified constitutive mouse liver NADPH-cytochrome P-450 reductase was successfully reconstituted in vitro with dilauroylphosphatidyl-choline and constitutive purified mouse testosterone 2 alpha-hydroxylase (cytochrome P-450(2)alpha) with an observed activity of 13.8 nmol.min-1.nmol P-450-1. Although the partially purified reductase obtained from the Fractogel column was contaminated by significant levels of two unidentified proteins, it was as equally effective in the reconstituted system as the DE-52-derived purified reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-450 reductases were similarly effective in supporting the catalytic activity of rat cytochrome P-450 2B1, but the murine reductase was 50% more effective than the rat reductase in a reconstituted system containing mouse cytochrome P-450(2)alpha.
Sharma
M C
MC
Laboratories of Biochemistry, University of Pennsylvania School, Philadelphia 19104-6048, USA.
Jeong
S J
SJ
Sharma
M R
MR
Shapiro
B H
BH
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001345:Autoradiography; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*; D051381:Rats; D013045:Species Specificity; D013250:Steroid Hydroxylases / Q000378:metabolism
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase