Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b(5) are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.
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.
Multiplication, the change of biologic properties with time, self-renewal—these are among the activities required of intracellular membranes in meeting the needs of the organism for growth, cellular specialization, and replacement. This article discusses current understanding of the dynamic molecular events that permit membranes to subserve and maintain their physiologic functions.
A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido-calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.
