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Mitochondrial NADH- or NADPH-Linked Aquacobalamin Reductase Activity Is Low in Human Skin Fibroblasts with Defects in Synthesis of Cobalamin Coenzymes¹

Fumio Watanabe², Hisako Saido^*, Ryoichi Yamaji^*, Kazutaka Miyatake^*, Yuji Isegawa, Akio Ito^**, Toshitsugu Yubisui, David S. Rosenblatt and Yoshihisa Nakano^*

Department of Food and Nutrition, Kochi Women's University, Kochi 780, Japan ^* Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Osaka 593, Japan Department of Preventive Medicine, Osaka University, Toyonaka, Osaka 560, Japan ^** Department of Biology, Kyushyu University, Fukuoka 812, Japan Department of Biology, Kochi University, Kochi 780, Japan MRC Genetics Group, Department of Medicine and Human Genetics, McGill University, Montreal, Quebec H3A 1A1, Canada

Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.

KEY WORDS: • vitamin B-12 • cobalamin • aquacobalamin reductase • human mutant fibroblasts • mitochondria

¹ 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.

² To whom correspondence should be addressed.

Manuscript received 21 May 1996. Revision accepted 2 August 1996.