Evidence for Molecular Identity of Microsomal and Mitochondrial NADH-Cytochrome b5 Reductases of Rat Liver

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Abstract
To confirm the molecular identity of the NADH-cytochrome b5 reductase of mitochondrial outer membrane with the corresponding enzyme of microsomes, the properties of mitochondrial and microsomal reductases were examined and compared. The NADH-cytochrome b5 reductase associated with rat liver mitochondria was easily solubilized by digestion with cathepsin D, exactly as the corresponding microsomal reductase was. The cytochrome b5-reducing activities of microsomal and mitochondrial reductases were both stimulated by about 80% upon solubilization from the membranes, whereas such stimulation was not observed when potassium ferricyanide was used as the electron acceptor. Some physicochemical properties and reaction kinetics of the solubilized microsomal and mitochondrial reductases were examined, and no significant differences were found between them. The rabbit antibody prepared against purified microsomal NADH-cytochrome b5 reductase inhibited the rotenone-insensitive NADH-cytochrome c reductase activities of microsomes and mitochondria to the same extent. The immunological identify of microsomal and mitochondrial reductases was confirmed by the Ouchterlony double diffusion test in agar gel and by quantitative immunoprecipitation tests using the cathepsin-solubilized enzyme preparations. The NADH-cytochrome b5 reductase molecules of mitochondrial outer membrane seem to be located on the outside surface of the membrane, since the antibody against the reductase inhibited the rotenone-insensitive NADH-cytochrome c reductase activity of intact mitochondria as well as that of hypotonic-treated mitochondria. The solubilization of the reductase from intact mitochondria by cathepsin D also supported this conclusion. It is thus concluded that the same reductase molecules are present on the cytoplasmic surfaces of endoplasmic reticulum and mitochondrial outer membrane in liver cells.

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