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.
Highlights
A priori, a complex membrane, like that of the hepatocyte endoplasmic reticulum, could be either (a) a homogeneous system in which all components, especially all constitutive enzymes, are randomly distributed, or (b) a mosaic in which each patch or tessera represents a specific group of enzymes and in which, all tesserae are not biochemically equivalent to one another
The results reported favor in part the same alternative, since the half-life of cytochrome bsturns. out to be significantly longer than that of NADPH-cytochrome c reductase, but the evidence cannot be considered conclusive because the half-life of the reductase is equal to the average halflife of all membrane proteins considered at two different levels of preparation, i.e. washed microsomes and residue of trypsin digestion
Since no evidence can be obtained at present on the other enzymes of the same electron transport chain, it cannot be decided whether the slower turnover of cytochrome bgis a characteristic of this individual protein or of the entire electron transport chain of which it is a part, and which could be visualized as a distinct tessera in a mosaic membrane
Summary
Deternzitition of Speci$c ilctivities of Proteins and Phospholipids of Rough and Smooth Membranes-In each experiment, 15 rats (150 to 200 g) fed ad lib&m a standard diet (Lab Chow, Ralston Purina Company) were used. Equal amounts of liver were removed from each of the three animals to give a final wet weight of 5 g. The pooled samples were homogenized in 0.25 M sucrose to give a final 25% (w/v) concentration. The homogenate was centrifuged at 10,000 X g (average) for 20 min to remove all particles larger than microsomes. A total microsome pellet (containing both rough and smooth microsomes) was obtained by diluting the ensuing supernatant solution 2.5 times with 0.25 M sucrose, and centrifuging at 105,000 X g for 180 min. The rough and smooth microsomes were separated by the method of Dallner et al [2, 3], which uses a discontinuous sucrose gradient, with homogenate in 0.25 M sucrose over 1.3 M sucrose, each made
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