scholarly journals Preparation and characterization of the plasma membrane of pig lymphocytes

1970 ◽  
Vol 120 (1) ◽  
pp. 133-143 ◽  
Author(s):  
D. Allan ◽  
M. J. Crumpton
Keyword(s):  
Plasma Membrane ◽  
Lymph Node ◽  
Membrane Fraction ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Whole Cells ◽  
Mesenteric Lymph ◽  
Specific Activities

Lymphocyte plasma membrane was isolated from minced pig mesenteric lymph node by differential centrifugation and by centrifuging through a sucrose density gradient. The yield of membrane was approx. 0.1% (dry wt. relative to wet wt. of lymph node). The purified material had a sucrose density of 1.14g/cm3 and consisted mainly of smooth vesicles. The membrane fraction contained, apart from protein and lipid, 59μg of carbohydrate, 11μg of sialic acid and 28μg of RNA/mg of protein; no DNA was detected. The cholesterol/phospholipid molar ratio was 1.01. Specific activities (μmol of product/h per mg of protein) of 5′-nucleotidase, succinate dehydrogenase, acid phosphatase and glucose 6-phosphatase were 10.1, 0, 0.51 and 0.30 respectively. The membrane vesicles were aggregated by an antiserum against pig lymphocytes and adsorbed the agglutinins to whole lymphocytes present in the antiserum; the membrane fraction was 28 times as effective as whole cells (on a dry wt. basis) in removing the lympho-agglutinins. Antisera against the membrane fraction agglutinated whole lymphocytes. It is concluded that the preparation represents the plasma membrane of small lymphocytes. The plasma membrane of pig thymocytes was isolated by using the same procedure. Its properties were similar to those of the lymphocyte plasma membrane.

Isolation and characterization of plasma membranes from bovine carotid arteries

1986 ◽  
Vol 250 (1) ◽  
pp. C65-C75 ◽  
Author(s):  
R. V. Sharma ◽  
R. C. Bhalla
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Carotid Arteries ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Plasma Membrane Fraction

A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.

scholarly journals ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

1969 ◽  
Vol 41 (2) ◽  
pp. 378-392 ◽  
Author(s):  
Charles W. Boone ◽  
Lincoln E. Ford ◽  
Howard E. Bond ◽  
Donald C. Stuart ◽  
Dianne Lorenz
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicle ◽  
Whole Cells ◽  
Continuous Sucrose Gradient

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

scholarly journals Carbohydrate composition of lymphocyte plasma membrane from pig mesenteric lymph node

1976 ◽  
Vol 153 (1) ◽  
pp. 75-78 ◽  
Author(s):  
D Snary ◽  
A K Allen ◽  
R A Faulkes ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Lymph Node ◽  
Mesenteric Lymph Node ◽  
Amino Sugar ◽  
Membrane Lipid ◽  
Molar Ratio ◽  
Neutral Sugar ◽  
Mesenteric Lymph ◽  
Lipid Fractions ◽  
Almost All

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.

scholarly journals Lipid composition of lymphocyte plasma membrane from pig mesenteric lymph node

1976 ◽  
Vol 156 (1) ◽  
pp. 103-110 ◽  
Author(s):  
G M Levis ◽  
G P Evangelatos ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Oleic Acid ◽  
Lymph Node ◽  
Mesenteric Lymph Node ◽  
Lipid Composition ◽  
Membrane Fraction ◽  
Neutral Lipids ◽  
Lipid Fraction ◽  
Mesenteric Lymph ◽  
Major Ganglioside

1. The lipid fraction of the plasma membrane of pig mesenteric lymph-node lymphocytes contained primarily (94%) neutral lipids and phospholipids in about equal weights. The remianing lipid comprised glycosphingolipids (1.8%), gangliosides (o.27%)and probably ceramides (1.3%). The major phospholipid was phosphatidylcholine (46% of the total), and mono- and tri-hexosylceramides accounted for 72% of the glycosphingolipids. Haematoside was distributed between the glycosphingolipid and ganglioside fractions. The major ganglioside was monosialoganglioside. About 90% of the sialic acid was N-glycollylated. 2. A comparision of the lipid composition of the plasma-membrane fraction with that of the initial lymph-node homogenate showed that the purified membrane contained increased proportions of phospholipids, especially sphingomyelin, glycosphingolipids and gangliosides. 3. Fatty acid analyses showed that the membrane phosphatidylcholine was rich in palmitic acid, that the sphingomeyelin and phosphatidylethanolamine were high in myristic acid and that the glycosphingolipids were rich in oleic acid.

scholarly journals Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 315-322 ◽  
Author(s):  
L Raeymaekers ◽  
F Wuytack ◽  
J Eggermont ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Membrane Vesicles ◽  
Cholesterol Content ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Fraction

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

scholarly journals Isolation and characterization of rabbit kidney brush borders

1972 ◽  
Vol 128 (5) ◽  
pp. 1319-1328 ◽  
Author(s):  
S. J. Quirk ◽  
G. B. Robinson
Keyword(s):  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Isolation And Characterization ◽  
Adenosine Triphosphatases ◽  
Brush Borders

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg2+-dependent and Na+,K+-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.

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