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At least 19 recordsLinked to original sources
PubMed1977

A novel system for the two-dimensional electrophoresis of membrane proteins.

Membrane proteins were resolved in two dimensions by a novel technique that uses discontinuous electrophoresis in both directions. After electrophoresis in the first direction in chloral hydrate, the membrane proteins were further resolved by a novel system that used organic-base dodecyl sulphates to stack and then resolve them. This latter system has several advantages over conventional electrophoresis in sodium dodecyl sulphate, notably that it avoids the production of artifacts generated by other systems.

Animals↗
PubMed1979

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.

Cholesterol↗
PubMed1976

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system.

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.

Binding Sites↗
PubMed1977

Assembly of viral membranes. I. Association of vesicular stomatitis virus membrane proteins and membranes in a cell-free system.

We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.

Cell Line↗
PubMed1977

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.

Adenosine Triphosphate↗
PubMed1977

Compositional homology of membrane-protein systems and membrane-associated proteins: comparison with milk fat globule membrane and "membrane"-derived xanthine oxidase.

Amino acid compositions of the milk fat globule membrane protein and plasma membrane protein from other sources, as well as the compositions of milk fat globule membrane-derived xanthine oxidase and selected plasma membranes-associated proteins were compared by statistical difference index. Additionally, the average hydrophobicity of xanthine oxidase and selected membrane proteins were compared. These comparisons indicate high orders of apparent compositional homology between the various plasma membranes and membrane-associated proteins. Because the biological functions of membrane proteins are widely diverse, it is speculated that their "relatedness" may reflect on evolutionary convergence to similar amino acid compositions, necessitated by their in situ environment--the lipoidal bilayer. However, compositional relatedness should not imply sequential homology.

Amino Acids↗
PubMed1979

Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.

Acetates↗
PubMed1979

Kinetics of red cell membrane phosphorylation: altered affinity of HS membrane protein acceptors.

Membrane ghosts were prepared from red blood cells of normal and hereditary spherocytosis (HS) subjects. Time dependent phosphorylation studies of the membrane (in the presence of gamma labelled AT32P) showed that the HS membrane appeared to incorporate less phosphate than the normal membrane but the results were not significantly different. Using the initial linear rate of phosphorylation and varying the ATP concentration, it was found that the Michaelis constant (Km) for the normal membrane was 24.8 microns ATP and Vmax was 0.120 nmol phosphate bound/mg membrane protein/min. These values for the HS membrane were found to be 39.0 and 0.118 respectively. In one family the affected mother and son each showed a positive cooperativity effect for similar reciprocal plots. This indicates that phosphorylation of various HS membrane proteins may be an ordered rather than random process and suggests biochemical heterogencity of the HS condition. The constants, Km and Vmax, however, were found to be similar to the rest of HS subjects studied. Crude extracts of the enzyme protein kinase, which catalyses the membrane phosphorylation, showed no significant difference in the Km and Vmax value in HS compared with that from normal red cells. The kinetic difference in phosphorylation is probably due to abnormalities in the membrane protein(s) which accept the phosphate from protein kinase in HS red cells.

Adenosine Triphosphate↗
PubMed1979

[Chromatography of membrane proteins in Triton-X 100. I) Solubilization of membrane proteins in Triton-X 100].

In their study of the chemical and physical characteristics of membrane protein, the Authors have directed their attention to Triton-X 100. The hypothesis is that this detergent would have two antagonistic effects: on the one hand it would delimit the membrane fostering the formation of unstable complexes, on the other it would establish unstable intermediate complexes fostering the formation of soluble proteic complexes.

Animals↗
PubMed1978

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes.

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.

Animals↗
PubMed1977

The structure of intrinsic membrane proteins.

Intrinsic membrane proteins are embedded in the lipid bilayer so that the polypeptides come in contact with the non-polar region of the bilayer. There are two major types of intrinsic proteins: those with most of their mass outside the cytoplasm (Type I) and those with most of their mass inside the cytoplasm (Type II). In the latter group are the membrane transport systems. The anion exchange system of the human erythrocyte is a dimer of band 3 polypeptides. These polypeptides span the bilary, have most of their mass in the cytoplasm, and are glycosylated. About 20-25% of the polypeptide, however, is in the bilayer. Arguments are presented to support the view that the intramembrane segments of the protein are alpha-helical and that the major protein-protein interactions between the subunits are in the cytoplasmic portion of the protein.

Biological Transport, Active↗
PubMed1979

[Chromatography of membrane proteins in Triton-X 100. II) Affinity chromatography of membrane proteins on Con-A linked to Sepharose in Triton-X 100].

The Authors have made chromatographs using Con-A Sefaroso columns, varying the percent of Triton-X 100 and the proteic concentration. With proteic concentrations varying from 0.4 to 4mg/ml and in solutions containing 0.5 to 1% of Triton-X 100 with the pH value remaining constant at 8 - solubilization of membrane protein does occur.

Animals↗
PubMed1978

Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm.

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.

Adenosine Triphosphatases↗
PubMed1976

Biochemical studies on abnormal erythrocyte membranes. Protein abnormality of erythrocyte membrane in biliary obstruction.

Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken, By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.

Cholestasis↗
PubMed1976

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver.

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...

Animals↗
PubMed1975

Immunochemical analyses of membrane-bound complement. Detection of the terminal complement complex and its similarity to "intrinsic" erythrocyte membrane proteins.

(1) Membranes of sheep erythrocytes lysed with antibody and human or rabbit complement were solubilized in non-ionic detergents (Triton X-100 or Berol EMU-043) and analysed immunochemically using antisera directed against individual complement components. The precipitation behaviour of membrane-bound C3, C5, C6 and C9 components of complement was examined by immuno-double diffusion, rocket- and crossed immunoelectrophoresis performed in agarose gels containing 1% non-ionic detergent. (2) Membrane-bound C5, C6 and C9 are antigenically altered compared with the native (serum) components. (3) Immuno-double diffusion in the presence of non-ionic detergents reveals formation of C5-C6-C9 complexes on the membranes; these complexes are stable in non-ionic detergent. No complex formation was detected in serum between native C5, C6 and C9 components. There was also no evidence for complexing between membrane-bound C3, C4 or membrane proteins and the "late-reacting" complement components. (4) The extractability of complement components by various manipulations has been studied by use of quantitative rocket immunoelectrophoresis. Up to 65% of membrane-bound C3 is readily extracted by dialysis of membranes against 1mM EDTA, pH 8.0, 100 mM EDTA, pH 8.0, 1.2 NaCl plus or minus EDTA, by extraction in isotonic buffers at 37 degrees C, by heating at 45 degrees C over several hours, or by treating membranes with 1 mM p-chloromercuribenzoate sulfonate. In contrast, less than 6% of the terminal complement complex can be eluted by any of the described methods or combination of methods. (5) Our data suggest that the terminal complement complex associates with membrane "core" components through apolar interactions.

Animals↗