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Mitochondrial NADPH-Linked Aquacobalamin Reductase Is Distinct from the NADPH-Linked Enzyme from Microsomal Membranes in Rat Liver^{1, 2,}

Hisako Saido, Fumio Watanabe^*, Yoshiyuki Tamura^*, Yoshihiko Funae, Susumu Imaoka and Yoshihisa Nakano³

Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 593 ^* Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, 1-89-1 Hamadera-minamimachi, Sakai, Osaka 592 Laboratory of Chemistry, Osaka City University Medical School, Osaka, 545, Japan

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 µmol·min^-1·mg protein^-1), potassium ferricyanide (4.9 µmol·min^-1·mg protein^-1) and 2,6-dichlorophenolindophenol (16.8 µmol·min^-1·mg protein^-1) as well as aquacobalamin (6.4 µmol·min^-1·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.

KEY WORDS: • cobalamin • aquacobalamin reductase • NADPH-cytochrome c reductase • rats

¹ Supported in part by a research grant from the Japanese Ministry of Education, Science and Culture (F. W.).

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

³ To whom correspondence should be addressed.

Manuscript received 23 April 1993. Revision accepted 1 July 1993.