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J. Biochem, 1970, Vol. 67, No. 2 199-210
© 1970 Japanese Biochemical Society

research-article

Vitamin K3-dependent NADPH Oxidase of Liver Microsomes

Purification, Properties, and Identity with Microsomal NADPH-cytochrome c Reductase

HIROKO NISHIBAYASHI-YAMASHITA and RYO SATO

Institute for Protein Research, Osaka University Kita-ku, Osaka

A flavoprotein catalyzing the oxidation of NADPH by oxygen in the presence of vitamin K3 or other appropriate naphthoquinones as cofactor was solubilized and purified from rabbit liver microsomes. The most purified preparation was homogeneous in both sedimentation and electrophoretic analyses. Its molecular weight was determined to be 89, 000 and two moles of FAD were present per mole of protein. The enzyme-bound flavin was reducible not only by NADPH but also by NADH. In addition to the naphthoquinone-dependent NADPH oxidase activity, the purified enzyme catalyzed the NADPH-linked reductions of a variety of acceptors such as benzoquinones, ferricyanide, redox dyes and cytochrome c. Cytochrome b3 was also reduced slowly. NADH could act as a weak electron donor in the K3-dependent oxidase activity. The effects of pH, ionic strength and various inhibitors on both the K3-dependent NADPH oxidase and NADPH-cytochrome c reductase activities were compared. The results of these studies as well as the finding that both activities were purified in parallel established the identity of K3-dependent NADPH oxidase with microsomal NADPH-cytochrome c reductase [EC 1.6.2.3 [EC] ]. Several kinetic parameters for the K3-dependent NADPH oxidase activity were determined. Finally, it was confirmed that in the oxidase reaction hydrogen peroxide was formed as the reduction product of oxygen.


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