Synthesis of cytochrome oxidase in isolated rat hepatocytes.

Abstract 1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharoseprotein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [ 35 S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase. 4. Antibodies to cytochrome oxidase specifically absorb two high molecular weight microsomal peptides which do not correspond to subunits of the holoenzyme. These peptides turnover rapidly after a pulse chase, but no concomitant increase in the cytoplasmically translated subunits were observed. A second band (50 000–55 000) was also absorbed from isolated microsomes, but in our hands is an artifact due to electrophoresis. Thus, we have not been able to identify a high molecular weight polyprotein precursor to the cytoplasmically translated subunits of cytochrome oxidase.