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Biomedical subjects

G Dallner

Publications and source records attributed to G Dallner.

At least 181 records · Page 10Linked to original sources
PubMed1979

Interaction of lectins with proteins of the endoplasmic reticulum and Golgi system of rat liver.

The interaction of glycoproteins of rough and smooth microsomal and Golgi membranes with Sepharose-bound lectins has been studied. One of these lectins was a crude preparation from wheat germ lipase which was found to bind primarily to N-acetyl neuraminic acid. Rough microsomes, smooth microsomes and Golgi membranes contain glycoproteins which bind to Concanavalin A (Con A specific for mannose residues) in decreasing amounts in the order indicated (rough, smooth and Golgi) and to wheat germ agglutinin (WGA, glucosamine-specific) and to the crude lipase preparation in increasing amounts in the order indicated. The small amount of binding of rough microsomes and Golgi membranes to Crotalaria (galactose-specific) increases substantially after neuraminidase treatment. Three submicrosomal particle preparations enriched either in AMPase or in NADH- or NADPH-oxidizing electron-transport enzymes contain glycoproteins which bind Con A and wheat germ agglutinin. The latter binding is sensitive to neuraminidase treatment. Two other submicrosomal particle preparations, both enriched in glucose-6-phosphatase activity, bind preferentially to WGA. This binding is, however, not sensitive to neuraminidase. Prolonged incubation with Ervilia lectin (mannose-specific) inhibits NADH-ferricyanide reductase activity, while the electron-transport chain involving cytochrome b5 is also inhibited by Crotalaria, indicating that both the flavoprotein and the cytochrome b5 are glycoproteins whose oligosaccharide chains have terminal mannose or galactose residues.

Animals↗
PubMed1978

Localization of protein-bound carbohydrate residues on the cytoplasmic surface of rough and smooth microsomes and Golgi vesicles from rat liver.

Rough and smooth microsomes and Golgi membranes isolated from rat liver were treated with proteolytic enzymes under conditions which removed 30--40% of the surface proteins without seriously disrupting the membrane structure. This treatment also removed 40--60% of protein-bound mannose, galactose and glucosamine. When protease treatment was combined with neuraminidase treatment, 80% of the sialic acid was removed from intact rough microsomal and Golgi vesicles and about half of the sialic acid of smooth microsomes was solubilized. It appears that half, or probably more, of the membrane glycoproteins are associated with the cytoplasmic surface of these membranes.

Animals↗
PubMed1978

Incorporation of galactose from UDP-galactose into microsomal and Golgi membranes of rat liver.

Rough and smooth microsomes and Golgi membranes were incubated with UDP[14C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from beta-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.

Animals↗
PubMed1978

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and Golgi membranes from rat liver.

Rough and smooth microsomes and Golgi membranes incorporate N-acetylglucosamine from UDP-N-acetylglucosamine into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possiblity that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.

Acetylglucosamine↗
PubMed1978

Mannosylation of endogenous proteins of rough and smooth endoplasmic reticulum and of Golgi membranes.

Mannosylation of the proteins of microsomal and Golgi membranes was investigated both after incubation in vitro of the isolated subfractions with GDP-[14C]mannose and after injection of [3H]mannose into rats followed by separation of these subfractions. Mannosylation of endogenous and added exogenous dolichol phosphate and also of dolichol pyrophosphate-oligosaccharide occurs in all three fractions. It was essential to inhibit antagonistic enzymes during incubation and to centrifuge after incubation. The presence of detergent in the incubation mixture influences the incorporation pattern of the different fractions in very different ways. In a system in vitro predominantly membrane proteins and not secretory proteins are mannosylated. Trypsin treatment of intact vesicles removes components from the outer surface only; such treatment liberates about one third of the radioactive mannose associated with lipid, releases radioactivity from the protein acceptor to the same extent and causes some inactivation of the transferase activities. It appears that a part of the mannosyl transferase system in rough and smooth endoplasmic reticulum and in Golgi membranes is localized at the cytoplasmic side of these membranes. This activity is probably involved in the glycosylation of proteins localized at the cytoplasmic surface of the endoplasmic reticulum.

Animals↗
PubMed1978

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate.

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b5, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.

Animals↗
PubMed1978

Glycosyl transfer from nucleotide sugars to C85- and C55-polyprenyl and retinyl phosphates by microsomal subfractions and Golgi membranes of rat liver.

The capacity of isolated membrane fractions to catalyse transfer of sugars from sugar nucleotides to alpha-saturated and non-saturated forms of phosphorylated C85 and C55 polyprenols and retinyl phosphate was examined. The amount of endogenous lipid acceptor present for various sugars was also measured. It appears that the types and amounts of polyprenyl phosphates present in rough- and smooth-microsomal fractions and Golgi membranes are different and the individual polyprenyl phosphates exhibit specificity as sugar acceptors.

Animals↗
PubMed1977

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver.

Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbital-treated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.

Animals↗
PubMed1977

Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.

Animals↗
PubMed1977

Transverse asymmetry of phospholipids in subcellular membranes of rat liver.

Subcellular membranes isolated from rat liver in a form impermeable to macromolecules were treated with phospholipase A2 from Naja naja venom. The phosphatidylserine, phosphatidylethanolamine and about half of the phosphatidylcholine of microsomes, Golgi membranes, inner mitochondrial membranes, lysosomes and nuclear membranes were hydrolyzed. It is proposed that these phospholipids are localized in the outer surface of the membrane bilayer, which represents the cytoplasmic side in the living cell, while the remaining phosphatidylcholine and most of the phosphatidylinositol, sphingomyelin and cardiolipin may be assigned to the inner side of the bilayer.

Animals↗
PubMed1977

Enzyme and phospholipid asymmetry in liver microsomal membranes.

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.

Adenosine Triphosphatases↗
PubMed1977

Electrophoretic mobility of microsomes from rat liver.

The electrophoretic mobilities of rough and smooth microsomes were studied using free electrophoresis in a sucrose gradient. Rough microsomes have a higher net negative surface charge but removal of the ribosomes decreases their mobility to that of smooth microsomes. Treatment with neuraminidase and phospholipases C and D does not affect the mobility of total smooth microsomes, but this mobility is increased by approximately 20% after trypsin and papain treatment and by approximately 12% after phospholipase A treatment. Further treatment of trypsin-digested smooth microsomes with phospholipase C re-establishes the original mobility. This effect is not caused by the removal of lipid phosphate groups, but by the liberation of negatively charged protein species that are normally buried under trypsin-sensitive proteins. Low concentrations of trypsin also solubilize enzyme proteins from smooth liver microsomes of phenobarbital-treated rats, but the electrophoretic mobility is not increased, indicating structural differences between induced and control membranes.

Animals↗
PubMed1976

Biogenesis of microsomal membrane glycoproteins in rat liver. IV. Characteristics of a cytoplasmic lipoprotein having properties of a membrane precursor.

The supernatant fraction from a rat liver homogenate contains a lipoprotein complex which can be incorporated into microsomal membranes in vitro. The lipoprotein can be purified by gel filtration and flotation in a NaBr solution. The isolated lipoprotein has an equilibrium density in the range of 1.07-1.14 g/ml, a sedimentation coefficient of 4.9 S and a calculated molecular weight of 210 000. The isolated complex contains 44% lipid by weight, half of which is phospholipid. The protein also has covalently bound sugar moieties, namely mannose, galactose, glucosamine, and sialic acid. The complex is unstable and may dissociate during the isolation procedure. The complex and its fragments contain two peptides with molecular weights of 11-13000 and 67-69000. These two components have practically identical amino acid compositions.

Amino Acids↗