Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.     

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

Summary The process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with¹²⁵I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.Abbrevations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid     

Antioxidants as cytochrome stabilizers in endoplasmic reticulum membranes of rat liver in vivo]

It has been shown that synthetic antioxidant 4-methyl-2, 6-ditrebuthylphenole (ionole) inhibits free radical oxidation of phospholipids in the membranes of endoplasmic reticulum, increases the life time of cytochrome P-448 after its induction with 20 methylcholantrene.     

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with -amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca²⁺. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.Abbreviations SER smooth endoplasmic reticulum - RER rough endoplasmic reticulum - PMS post mitochondrial supernatant - MES 2-(N-morpholino) ethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Composition ofNeurospora crassa vacuolar membranes and comparison to endoplasmic reticulum,plasma membranes,and mitochondrial membranes

We have determined the carbohydrate and lipid contents of vacuolar membranes fromNeurospora crassa, and have compared them to mitochondrial membranes, endoplasmic reticulum, and plasma membranes. These four membrane fractions were clearly distinct from each other in polypeptide composition, as judged by polyacrylamide gel electrophoresis. The vacuolar membranes proved unusual in two respects: the contained very high amounts of carbohydrate and were the only membranes with significant levels of phosphatidylserine. As in other eucaryotic cells, the mitochondrial membranes were unique in having high amounts of cardiolipin but virtually no sterol. Although the endoplasmic reticulum and plasma membranes were qualitatively similar to each other, the plasma membranes could be distinguished by a higher carbohydrate content, whereas the endoplasmic reticulum had a characteristically high ratio of phosphatidylcholine to phosphatidylethanolamine. The fatty acid compositions of all four membranes were similar, except that mitochondrial membranes contained about half as much saturated fatty acids as the other three fractions.     

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Summary The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradients as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0°C; previous treatment with LiCl does not modify the behavior of S.E>R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

Glycine and serine rich peptides isolated from rat liver preparations enriched with smooth endoplasmic membranes

Three electrochromatographic homogeneous peptides belonging to acidic, basic and neutral electrophoretic glycopeptide fractions isolated from rat liver preparations enriched with smooth endoplasmic membranes contain a very high level of glycine and serine and only a slight level of phenylalanine but do not contain tyrosine. Their amino acid compositions differ chiefly in the contents of basic and acidic amino acids.     

Biosynthesis of sphingomyelin in rat liver endoplasmic reticulum.

Rat liver microsomal sphingomyelin synthetase (CDPcholine: N-acylspingosine choline phosphotransferase (EC 2.7.8.3)) has been shown to be markedly stimulated by ATP and pantothenic acid derivatives such as CoA, pantethine, pantetheine and 4'-phosphopantetheine.     

Functional changes of endoplasmic reticulum membranes associated with liver regeneration

The fluidity and lipid composition of microsomal membranes have been studied at the earliest stage of liver regeneration in the rat (16 h after partial hepatectomy). The physical properties of the membranes have been measured by electron paramagnetic resonance analysis of freedom of motion of lipid and protein analogue probes. The fluidity of the hydrophobic core and of the microenvironment surrounding membrane proteins appeared to be modified, while no modifications were detectable in the fluidity at the surface or in bulk biochemical composition. The kinetic parameters of two enzymes of the endoplasmic reticulum (3-hydroxy-3-methyl glutaryl coenzyme A reductase and glucose-6-phosphatase) which are differentially localized within the membrane bilayer, were also measured. The temperature dependence of both enzymes was modified in the proliferating system, but these modifications were not consistent with the changes detectable in their specific activity. A model to explain the changes that occur in this proliferating membrane system is presented.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.