Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.
NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4).
Menting
J G
JG
Department of Biochemistry, La Trobe University, Victoria, Australia.
Cornish
E
E
Scopes
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011817:Rabbits / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D009251:NADPH-Ferrihemoprotein Reductase