Protein synthesis in mitochondria. 3. The controlled disruption and subfractionation of mitochondria labelled in vitro with radioactive valine

Abstract

Rat-liver mitochondria were incubated in vitro with radioactive valine and the distribution of radioactive protein was determined in mito-chondrial subfractions obtained after disruption with detergent. The extent of disruption by Triton detergents was affected by the chain length of the detergent, the detergent concentration, the mitochondrial concentration and the time of their exposure to detergent. By variation of these factors it was possible to obtain controlled disruption and to extract the various components of mitochondria in a sequential fashion. By differential centrifugation of the disrupted mitochondria, a fraction rich in respiratory enzymes, ribonucleic acid and phospholipid, and containing the bulk of the radioactive protein, was obtained. The fraction was probably derived from the mitochondrial membrane. Treatment of this fraction with more detergent separated it into a ribonucleic acid-rich component and a lipid-rich component, the latter having protein of higher specific radioactivity. Treatment of the crude lipoprotein in the lipid-rich component with butan-1-ol gave a protein similar in properties to the "structural protein" of ox-heart mitochondria described by other workers. Fractionation of the lipid-rich component on calcium phosphate revealed the presence of at least 2 radioactive proteins. It is concluded that the major site of incorporation of amino acids, in vitro, is into insoluble lipoprotein, probably derived from the mitochondrial membrane. An hypothesis to explain the pattern of labelling observed in vitro is presented.