PURIFICATION AND PARTIAL CHARACTERIZATION OF NADPH-CYTOCHROME-C REDUCTASE FROM PETUNIA-HYBRIDA FLOWERS

NADPH-cytochrome c reductase was solubilized from the microsomal fract ion of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammoni o]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisph osphate-Sepharose chromatography, followed by high-performance anion-e xchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfat e-polyacrylamide gel electrophoresis. Western blot analysis showed tha t both purified proteins cross-reacted with two different monoclonal a ntibodies raised against P. hybrida NADPH-cytochrome c reductase and r abbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibod ies. Only one 84-kD protein was detected by western blot analysis of f resh microsomal extracts. Amino acid sequence analysis of tryptic pept ides revealed significant similarity to the NADPH binding region of pl ant and animal NADPH-cytochrome P450 reductases and Bacillus megateriu m cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the K-m values for the bind ing of NADPH and ferricytochrome c were 9.2 and 2.8 mu M, respectively . We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4).