Purification and characterization of cytochrome P450 reductase from liver microsomes of feral leaping mullet (Liza saliens).
NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 mumol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 +/- 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent K(m) of the purified enzyme was found to be 7.69 microM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 microM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties.
Sen
A
A
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
Arinc
E
E
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification* / Q000378:metabolism; D010473:Perciformes / Q000378:metabolism*
D009251:NADPH-Ferrihemoprotein Reductase