Turnover of Constituents of the Endoplasmic Reticulum Membranes of Rat Hepatocytes

Abstract

Abstract The turnover of various constituents of the membranes of the endoplasmic reticulum has been measured by injecting 14C-leucine and 14C-acetate, or in some cases 14C-leucine and 14C-glycerol, into a series of 12 to 15 rats (weighing 150 to 200 g). At various times, up to 2 weeks after injection, three rats were killed, their livers were pooled, microsomes were obtained and divided into rough and smooth fractions, and purified membranes were isolated from each fraction. The constituents examined were the following: total membrane proteins; two specific membrane components, purified reduced-NADP: ferricytochrome c oxidoreductase and purified cytochrome b5; total membrane lipids; and the nonpolar (fatty acid) and polar (glycerol backbone) moieties of the lipid. The two enzymes were purified 100- to 150-fold by trypsin digestion of KCl-washed microsomes, followed by chromatography on Sephadex G-100 and diethylaminoethyl cellulose. The purity of both proteins was close to 100%, in yields of 25 to 35% from original microsomes. Both total proteins and total lipids of rough microsomal membranes had the same half-lives as their counterparts from smooth microsomal membranes. In the various experiments, the total membrane proteins had half-lives of from 75 to 113 hours, while in the same experiments, total membrane lipids have 10 to 30% shorter half-lives. The half-life of the NADPH-cytochrome c reductase was, in two experiments, almost exactly that of total membrane proteins, while the half-life of cytochrome b5 was significantly (about 50%) longer. The polar and nonpolar components of the lipids had different apparent half-lives, that of fatty acids being much longer than that of the glycerol backbone. The longer half-life of fatty acids may be connected with the presumed presence of transacylating enzymes in microsomal membranes. The results are discussed in relation to current concepts on membrane structure and biogenesis.