Structural studies of purified pig lymph node plasma membrane: association of cytoskeletal components with the plasma membrane and the effect of detergent solubilization

1982 ◽  
Vol 60 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Robert J. Allore ◽  
Brian H. Barber
Keyword(s):  
Plasma Membrane ◽  
Lymph Node ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Sodium Deoxycholate ◽  
Insoluble Fraction ◽  
Plasma Membrane Vesicles ◽  
Detergent Solubilization ◽  
Lentil Lectin

The reproducibility of preparation, stability at 4 °C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found mat 2% (w/v) Nonidet P-40 solubilized 50–60% and 2% (w/v) sodium deoxycholate solubilized 60–70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate – polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet P-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found mat very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.

Electroimmunochemical analysis of plasma membrane vesicles from Saccharomyces cerevisiae

1982 ◽  
Vol 60 (6) ◽  
pp. 659-667
Author(s):  
James H. Gerlach ◽  
Ole J. Bjerrum ◽  
Gerald H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Concanavalin A ◽  
Periodic Acid ◽  
Membrane Vesicles ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Crossed Immunoelectrophoresis ◽  
Lentil Lectin ◽  
Triton X

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly.Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid – Schiff procedure. None of the glycoproteins had their origin in the cell wall.Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins.Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes

1977 ◽  
Vol 163 (2) ◽  
pp. 211-217 ◽  
Author(s):  
I S Trowbridge ◽  
M Nilsen-Hamilton ◽  
R T Hamilton ◽  
M J Bevan
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
Sodium Deoxycholate ◽  
Pea Lectin ◽  
Preliminary Characterization ◽  
Low Concentrations ◽  
Membrane Bound

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.

Reconstitution of D-glucose transporter from human placental microvillous plasma membranes

1982 ◽  
Vol 242 (3) ◽  
pp. C166-C171 ◽  
Author(s):  
J. M. Bissonnette ◽  
J. A. Black ◽  
K. L. Thornburg ◽  
K. M. Acott ◽  
P. L. Koch
Keyword(s):  
Glucose Uptake ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Human Trophoblast

Proteins from microvillous plasma membrane vesicles of the maternal surface of human trophoblast were solubilized with octyl beta-D-glucoside. After incorporation of the soluble protein into phospholipid liposomes D-glucose uptake exceeded that of L-glucose. The reconstituted system showed that D-glucose uptake was not sodium dependent and was inhibited by cytochalasin B. Efflux of D-glucose from the proteoliposomes was retarded by cytochalasin B. D-Glucose uptake, but not L-glucose, was proportional to the amount of protein used in the reconstitution procedure. Membrane protein was also solubilized with octylglucoside from vesicles that had been extracted previously by dimethylmaleic anhydride. Proteoliposomes prepared from these latter proteins showed D-glucose uptake threefold greater than that from octylglucoside solubilization alone, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted protein showed no clear difference between the double extraction procedure and the pattern obtained with the single detergent.

Hexose transport in plasma membrane vesicles of rat myoblast L6

1984 ◽  
Vol 62 (11) ◽  
pp. 1217-1227 ◽  
Author(s):  
Matthias O. Cheung ◽  
Theodore C. Y. Lo
Keyword(s):  
Plasma Membrane ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Hexose Transport ◽  
Plasma Membrane Vesicles ◽  
Transport Studies ◽  
Ethanesulfonic Acid ◽  
Flow Dialysis

To determine the molecular mechanism of hexose transport in rat myoblasts, transport studies were carried out with purified plasma membrane vesicles. Rat myoblasts were homogenized and fractionated by differential and sucrose gradient centrifugation. Six different fractions were obtained. Studies with marker enzymes revealed that two fractions (A and B) were composed of only plasma membrane. These two fractions differed considerably in their physical properties. Fraction A was composed of large multilaminated vesicles, with an intravesicular volume of 50 μL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 μL/mg protein. Based on the response of the ouabain-sensitive Na+,K+-ATPase activity to sodium dodecyl sulfate and ionophore treatments, it seemed likely that fraction A was composed of a significant amount of sealed right-side-out vesicles, whereas fraction B was composed of mainly membrane sheets or leaky vesicles. The initial rate of hexose influx into the membrane vesicles was determined by the flow dialysis technique. The optimal conditions for 2-deoxyglucose (2-DG) uptake into the plasma membrane vesicles were either 50 mM phosphate buffer or 10 mM 2-(N-2-hydroxyethylpiperazin-N′-yl)ethanesulfonic acid buffer at pH 7.0. In the presence of 500 μM 2-DG, the initial rates of 2-DG influx were 295 and 49 nmol/min per milligram protein for fractions A and B, respectively. In other words, after 1 min of incubation, the intravesicular concentration of 2-DG was around 6 mM, about 10 times the extravesicular concentration. D-Glucose was taken up to a similar extent (333 nmol/min per milligram protein), whereas L-glucose only equilibrated across the plasma membrane. Analysis of the fate of 2-DG revealed that the substrate was not phosphorylated upon incubation with the vesicles. Transport activity can be abolished either by disruption of the membrane vesicles or by reduction of the electrical potential across the membrane.

scholarly journals Interactions between platelet-surface glycoproteins. Identification of glycoproteins capable of binding to platelet surfaces

1982 ◽  
Vol 202 (3) ◽  
pp. 707-716 ◽  
Author(s):  
D J Bowles ◽  
C Brunton
Keyword(s):  
Plasma Membrane ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Galactose Oxidase ◽  
Peanut Agglutinin ◽  
Platelet Surface ◽  
Lentil Lectin

1. Platelets have been isolated from plasma and their surface glycoconjugates radioactively-labelled using galactose oxidase and NaB3H4. 2. Conditions have been defined for optimal labelling of glycoproteins and a membrane fraction enriched in plasma membrane has been prepared and characterized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Desialylated glycoproteins that act as receptors to peanut agglutinin and lentil lectin have been purified from a detergent extract of plasma membrane. 4. Two glycosylated polypeptides that are able to bind to the surfaces of platelets have been identified and some characteristics of the binding have been investigated.

scholarly journals Electrophoretic characterization of hepatic alkaline phosphatase released by phosphatidylinositol-specific phospholipase C. A comparison with liver membrane and serum-soluble forms

1985 ◽  
Vol 227 (1) ◽  
pp. 183-189 ◽  
Author(s):  
T Kominami ◽  
A Miki ◽  
Y Ikehara
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phospholipase C ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Polyacrylamide Gel ◽  
Soluble Form ◽  
Enzyme Preparations

Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane.

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