Occurrence of Different Types of Cytochrome b5-like Hemoprotein in Liver Mitochondria and Their Intramitochondrial Localization

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Abstract
Hypotonic treatment of rat liver mitochondria caused the solubilization of a b-type cytochrome (“IS-cytochrome”) which was spectrally similar to cytochrome b5 and reducible by NADH in the presence of NADH-cytochrome b5 reductase [EC 1. 6. 2. 2]. Tryptic digestion of hypotonically treated mitochondria resulted in the release of another b5-like cytochrome (“OM-cytochrome”) into solution. Cell fractionation studies indicated that IS-cytochrome is a true mitochondrial constituent. Its solubilization behavior paralleled that of adenylate kinase [EC 2. 7. 4. 3], suggesting that it is located in the mitochondrial intermembrane space. Hypotonic solubilization of IS-cytochrome was also observed with mitochondria from rabbit liver, rabbit kidney and pig heart. The outer membrane purified from hypotonically treated rat liver mitochondria contained a high concentration of a b5-like cytochrome. This cytochrome could be solubilized by tryptic digestion of intact mitochondria in an isotonic medium. When exposed to a hypotonic medium, the trypsin-treated mitochondria could still release-IS-cytochrome, and the outer membrane isolated from these mitochondria was essentially free from b5-like cytochrome. It was thus suggested that OM-cytochrome is firmly bound to the outer surface of outer mitochondrial membrane. By Sephadex gel filtration IS-cytochrome was separated into two hemoprotein components, both having almost the same spectral properties. The large component (molecular weight, ∼120,000) was labile and converted gradually to the small one (molecular weight, ∼12,000) on incubation of mitochondria in the hypotonic medium. Although both components could reconstitute an NADH-cytochrome c reductase system when coupled with microsomal NADH-cytochrome b5 reductase, their efficiencies in this system were much lower than that of cytochrome b5. Neither of the IS-cytochrome components reacted with an antibody to rat liver microsomal cytochrome b5.

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