Purification and characterization of NADPH-dependent methemoglobin reductase from the nucleated erythrocytes of bullfrog, Rana catesbeiana.
Two protein components having a NADPH-dependent methemoglobin reductase activity were purified to electrophoretic homogeneity from the erythrocytes of the bullfrog, Rana catesbeiana. Their molecular properties were investigated. The components were separated by isoelectric focusing, having discrete bands of pI 5.0 and 7.5, respectively. The pI 5.0 component, designated F-5.0, was faint yellow, with a broad absorption in the range of 400-450 nm, while the pI 7.5 component, designated F-7.5, was colorless and did not absorb in that range. The molecular weight was estimated to be 22,000 for both components by gel filtration and SDS-PAGE. When F-5.0 was subjected to isoelectric focusing repeatedly, the protein part of that component gradually moved to and refocused at pH 7.5, leaving a yellow color at acidic pH. Both F-5.0 and F-7.5 were highly specific for NADPH and had the same kinetic properties in catalyzing the reduction of MB, DCPIP, FMN, or FAD, and that of methemoglobin or cytochrome c in the presence of a certain dye. They were also indistinguishable from one another in their amino acid compositions and were completely identical in the N-terminal sequence of 24 amino acid residues. These findings strongly suggest that the two components can be attributed to the same enzyme molecule, carrying an identical protein moiety but interacting differently with some unidentified biological pigments, and that they are equivalent in their molecular and kinetic properties to the NADPH-dependent enzyme(s) occurring in human erythrocytes.
Abe
Y
Y
Department of Biochemistry, Nippon Medical School, Tokyo.
Ito
T
T
Okazaki
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D011892:Rana catesbeiana
D000596:Amino Acids; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D009249:NADP; D042966:Cytochrome-B(5) Reductase; D009252:NADPH Dehydrogenase