Mitochondrial NADPH-linked aquacobalamin reductase is distinct from the NADPH-linked enzyme from microsomal membranes in rat liver.
Mitochondrial NADPH-linked aquacobalamin reductase was purified and characterized to clarify its enzymatic properties. The enzyme was purified about 360-fold over rat liver mitochondrial membranes in a yield of 7.5%. The purified enzyme was homogenous in SDS-PAGE. The molecular mass (M(r)) of the enzyme was calculated to be 65 kDa by SDS-PAGE and by Toyopearl HW55 gel filtration, indicating that the enzyme is a monomeric polypeptide with M(r) of 65 kDa. The enzyme was a flavoprotein containing 1 mol of FAD and FMN per mole of the enzyme. The enzyme was specific for NADPH as electron donor and had the ability to reduce cytochrome c (15.4 mumol.min-1 x mg protein-1), potassium ferricyanide (4.9 mumol.min-1 x mg protein-1) and 2,6-dichlorophenolindophenol (16.8 mumol.min-1.mg protein-1) as well as aquacobalamin (6.4 mumol.min-1 x mg protein-1). Although the enzyme immunoreacted with an antibody against NADPH-cytochrome P-450 reductase, which had the activity of the NADPH-linked aquacobalamin reductase in rat liver microsomes, the mitochondrial enzyme and the microsomal enzyme had different enzymological properties.
Saido
H
H
Department of Agricultural Chemistry, University of Osaka Prefecture, Japan.
Watanabe
F
F
Tamura
Y
Y
Funae
Y
Y
Imaoka
S
S
Nakano
Y
Y
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000032:analysis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D005415:Flavins; D011506:Proteins; C068157:aquacobalamin reductase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital