Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans.
Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11alpha position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11alpha-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c(3+). Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.
Makovec
Tomaz
T
Institute of Biochemistry, Medical faculty, University of Ljubljana, Vrazov trg 2, Slovenia.
Breskvar
Katja
K
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D012233:Rhizopus / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C074514:steroid 11-alpha-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin