Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Abstract

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome‐c‐Sepharose 4B or by chromatography on DEAE‐Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher V_max for the enzyme isolated by chromatography on DEAE‐Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme‐linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase‐conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I–III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.