Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis.
Euglena aquacobalamin reductase (NADPH: EC 1.6.99.-) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conversion of hydroxocobalamin to 5'-deoxyadenosylcobalamin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mitochondrial extract in a yield of 38%. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent Km of 43 microM and to hydroxocobalamin with an apparent Km of 55 microM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 degrees C, respectively.
D000818:Animals; D002850:Chromatography, Gel; D003038:Cobamides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005056:Euglena gracilis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006879:Hydroxocobalamin / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D013696:Temperature; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism
D003038:Cobamides; C043960:aquacobalamin; D009249:NADP; C053436:aquacobalamin reductase (NADPH); D009247:NADH, NADPH Oxidoreductases; C000913:cobamamide; D014805:Vitamin B 12; D006879:Hydroxocobalamin