Studies in Vitro on Amino Acid Incorporation into Purified Components of Rat Liver Mitochondria

Abstract

Abstract Several major protein components of the inner membrane were isolated from rat liver mitochondria after an incubation with (14C)leucine. No significant radioactivity was present in either the fraction containing the oligomycin-sensitive ATPase or that containing cytochromes b and c1. A variable amount of radioactive label was associated with a membranous preparation of cytochrome oxidase. After purification of this preparation to yield a highly purified and soluble cytochrome oxidase, however, a loss of radioactivity was observed. No counts were present in the two major bands observed after disc electrophoresis of the purified cytochrome oxidase. Three different methods were used to prepare a protein fraction which, in all three cases, appeared identical on polyacrylamide gel electrophoresis. The two structural protein fractions prepared with the use of dilute acid were not labeled, while the structural protein fraction prepared with bile salts and ammonium sulfate fractionation contained 40% of the total counts of the intact mitochondria. The radioactivity present in the latter structural protein fraction was not, however, present in any of the major bands visualized after disc electrophoresis but was retained with the small amount of material at the origin of the gel. It would thus appear that rat liver mitochondria in vitro do not incorporate amino acids into the major proteins of the structural protein fraction, cytochromes b and c1, cytochrome oxidase, or oligomycin-sensitive ATPase. Radioactive amino acids are incorporated in vitro into a heterogeneous insoluble membranous fraction which contains less than 10% of the total mitochondrial protein. The radioactivity in this fraction is associated to a significant degree only with the two slowest moving bands observed on disc electrophoresis plus the material retained at the origin of the gel. These results suggest that the proteins labeled by rat liver mitochondria in vitro are among the largest and least soluble of those in the mitochondrion.