scholarly journals Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets.

1976 ◽  
Vol 71 (2) ◽  
pp. 606-623 ◽  
Author(s):  
A Lernmark ◽  
A Nathans ◽  
D F Steiner
Keyword(s):  
Plasma Membrane ◽  
Wheat Germ Agglutinin ◽  
Membrane Fraction ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasma Membrane Fraction ◽  
Fresh Plasma ◽  
Rat Islets Of Langerhans ◽  
Membrane Components

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

scholarly journals Specific glycoprotein changes during development of the chick neural retina.

1978 ◽  
Vol 79 (1) ◽  
pp. 132-137 ◽  
Author(s):  
G Mintz ◽  
L Glaser
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Neural Retina ◽  
Cell Type ◽  
Retinal Antigens ◽  
Qualitative Changes

After separation of whole proteins of chick neural retina by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a number of glycoproteins can be detected by staining the gels with 125I-labeled wheat germ agglutinin (WGA) and other lectins. The glycoprotein patterns show both quantitative and qualitative changes between days 7 and 13 of development. Some of these glycoproteins can be separated by chromatography on columns of insolubilized lectins. These observations suggest that purification of some of these glycoproteins identified by staining with radioactive lectins would yield retinal antigens which may be specific for developmental stage and cell type.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

scholarly journals Further characterization of HeLa S3 plasma membrane ghosts

1978 ◽  
Vol 77 (2) ◽  
pp. 448-463 ◽  
Author(s):  
E Costantino-Ceccarini ◽  
PM Novikoff ◽  
PH Atkinson ◽  
AB Novikoff
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Simple Procedure ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Major Band ◽  
Plasma Membrane Fraction ◽  
Coomassie Blue ◽  
Hela S3

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.

scholarly journals Albumin associated with purified pig lymphocyte plasma membrane

1980 ◽  
Vol 192 (1) ◽  
pp. 49-57 ◽  
Author(s):  
M J Owen ◽  
B H Barber ◽  
R A Faulkes ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Serum Albumin ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Conditions ◽  
Isoelectric Points ◽  
Pig Serum

Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar “fingerprints” of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.

scholarly journals Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane

1976 ◽  
Vol 159 (2) ◽  
pp. 293-299 ◽  
Author(s):  
E R Abney ◽  
W H Evans ◽  
R M E. Parkhouse
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Mouse Spleen ◽  
Plasma Membrane Fraction ◽  
Nucleotide Pyrophosphatase ◽  
Outer Aspect ◽  
Pyrophosphatase Activity ◽  
Spleen Lymphocytes

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.

Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

1978 ◽  
Vol 56 (7) ◽  
pp. 713-721 ◽  
Author(s):  
I. M. Yousef ◽  
R. K. Murray
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Canalicular Membrane ◽  
Polypeptide Patterns ◽  
Cell Plasma ◽  
Bile Canalicular Membrane ◽  
And Storage

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3–CaCl2, and K2HPO4–KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3–CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (> 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to actin were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris–HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3–CaCl2 appeared to be the best buffer and Tris–HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

1990 ◽  
Vol 68 (4) ◽  
pp. 699-704 ◽  
Author(s):  
Yuzuru Kubohara ◽  
Koji Okamoto
Keyword(s):  
Dictyostelium Discoideum ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Stalk Cell ◽  
Affinity Column ◽  
Western Blots

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5–6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.Key words: Dictyostelium discoideum, Dictyostelium mucoroides, wheat germ agglutinin.

Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles

1991 ◽  
Vol 69 (4) ◽  
pp. 282-290 ◽  
Author(s):  
Darren D. Browning ◽  
Danton H. O'Day
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Fusion ◽  
Concanavalin A ◽  
Calcium Ions ◽  
Sexual Development ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dictyostelium Cell

To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase–antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 μg/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in D. discoideum.Key words: Dictyostelium, cell fusion, glycoprotein, tunicamycin, concanavalin A, wheat germ agglutinin, calcium.

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.

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