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     

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.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

GTP hydrolysis by transitional endoplasmic reticulum from rat liver inhibited by all-trans-retinol

GTP hydrolysis by an endoplasmic reticulum fraction from rat liver enriched in part-rough, part-smooth transition elements was inhibited by all-trans-retinol half maximally at a concentration of about 10 micrograms/ml. Similar results were obtained with GTPase activity partially purified by ion-exchange (DE-52) chromatography. The inhibition was non-competitive and given by both retinol and retinaldehyde but not by retinoic acid or alpha-tocopheryl acetate. The hydrolysis of other nucleoside di- and triphosphates was much less affected by retinol. The activity was inhibited by detergents but at much higher concentrations than by retinol. The results suggest that enhancement of cell-free transfer from endoplasmic reticulum to Golgi apparatus by retinol observed previously at low concentrations of cytosol may be mediated through an interaction with GTP.