Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.
A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular  weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.
D000818:Animals; D000906:Antibodies; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000302:isolation & purification*; D007118:Immunoassay; D005779:Immunodiffusion; D007163:Immunosorbent Techniques / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D014176:Protein Biosynthesis; D051381:Rats
D000906:Antibodies; D004792:Enzyme Precursors; D003576:Electron Transport Complex IV