scholarly journals Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity

1982 ◽  
Vol 204 (1) ◽  
pp. 209-219 ◽  
Author(s):  
D R Critchley ◽  
C H Streuli ◽  
S Kellie ◽  
S Ansell ◽  
B Patel
Keyword(s):  
Cholera Toxin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activity ◽  
Galactose Oxidase ◽  
3T3 Cells ◽  
Ganglioside Gm1 ◽  
Intact Cells ◽  
Terminal Galactose ◽  
Toxin Receptor

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

Membrane Glycoprotein Defects In Bernard-Soulier Syndrome Platelets

1981 ◽  
Author(s):  
K J Clemetson ◽  
J L McGregor ◽  
E James ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Galactose Oxidase ◽  
Low Molecular Weight ◽  
Membrane Glycoprotein ◽  
One Dimensional ◽  
The Third ◽  
Healthy Donors ◽  
Bernard Soulier Syndrome

It is well established that Bernard-Soulier syndrome (BSS) platelets are deficient in a major membrane glycoprotein (lb). In order to investigate if this is the only defect in this disorder and to see if the β-subunit of glycoprotein lb is also diminished, platelets from 3 BSS patients and from healthy donors were isolated, washed and surface labelled by lactoperoxidase-catalysed iodination, pèriodate/NaB3H4 or neuraminidase/ galactose oxidase/NaB3H4. Labelled platelets were solubilized in sodium dodecyl sulphate and separated by 2-dimensional gel electrophoresis (isoelectric focusing, discontinuous polyacrylamide gel electrophoresis). Glycoprotein Ibα was virtually absent in 2 patients and strongly decreased in the third patient. The 3-subunit was also absent in the 2 patients and present at about 40 % of normal in the third. Glycoprotein IIbβ was present normally in all patients. In addition, a further low molecular weight glycoprotein with a M.WT. of 17,000 and a pI of 6.8-7.5 was absent or present at levels paralleling glycoprotein Ibβ. The thrombin cleavable glycoprotein (GP IV or V) appeared greatly diminished with BSS platelets labelled by carbo-hydrate specific methods though no difference could be seen with iodination. This finding was confirmed in a fourth BSS patient using one dimensional gel electrophoresis.The defects in BSS platelets are thus more complex than previously thought.

Unique T Cell Differentiation Markers: Gangliosides with Cholera Toxin Receptor Activity on Murine Fetal Thymocytes

1994 ◽  
Vol 156 (2) ◽  
pp. 402-413 ◽  
Author(s):  
Masaaki Noguchi ◽  
Zhang Suping ◽  
Junko Taguchi ◽  
Takao Hirano ◽  
Hiroshi Hashimoto ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Cholera Toxin ◽  
T Cell Differentiation ◽  
Receptor Activity ◽  
Differentiation Markers ◽  
Toxin Receptor

Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone

1989 ◽  
Vol 3 (3) ◽  
pp. 239-245 ◽  
Author(s):  
E. Uchida ◽  
T. Hayakawa ◽  
S. Niimi ◽  
A. Tanaka ◽  
M. Morikawa
Keyword(s):  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Type ◽  
3T3 Cells ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Gh Receptor ◽  
Adipose Conversion

ABSTRACT Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130 000 was observed along with minor complexes (Mr 300 000, 230 000 and 60 000) on autoradiography. Non-reducing—reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130 000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.

scholarly journals Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 255-260 ◽  
Author(s):  
K Suyama ◽  
J Goldstein
Keyword(s):  
Phospholipase A2 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cellular Membrane ◽  
Cytoplasmic Domain ◽  
Red Cell Membrane ◽  
Intact Cells ◽  
Antibody Preparation ◽  
D Cells ◽  
Papain Digestion

Abstract Intact erythrocytes of different Rh genotypes were subjected to various enzyme treatments, the effects of which were monitored by separating the membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and performing Western blotting using an antibody preparation that recognizes only Rh-related polypeptides. We found that treatment of intact cells with either phospholipase A2 or proteases such as papain did not alter the size of Rh antigen-containing polypeptides. In contrast, phospholipase A2 treatment followed by papain digestion cleaved a fraction of these polypeptides. This cleavage appears, from such digestions of Rh(D) positive and negative cells of different genotypes, to occur solely at the extracellular domain of Rh(D) polypeptide, while the extracellular domains of other Rh antigen-containing polypeptides are unaffected. Digestion of red blood cell ghosts and inside-out vesicles with trypsin showed that Rh(D), (C/c), and (E/e) antigen-containing polypeptides span the lipid bilayer having cytoplasmic domains susceptible to the action of proteases. The size of the cleavage products at the cytoplasmic domain of -D-/-D- cells was found to differ from that of other Rh(D) positive genotypes, due possibly to a difference in folding of Rh(D) polypeptide at its cytoplasmic domain and within the cellular membrane of these cells.

scholarly journals Platelet membrane studies in the May-Hegglin anomaly

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 279-284 ◽  
Author(s):  
BS Coller ◽  
MH Zarrabi
Keyword(s):  
Sialic Acid ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Glycoprotein ◽  
Galactose Oxidase ◽  
Periodic Acid Schiff ◽  
Giant Platelets ◽  
Total Sialic Acid ◽  
Bernard Soulier Syndrome

Abstract Since studies of the giant platelets in the Bernard-Soulier syndrome have shown decreased electrophoretic mobility, decreased sialic acid, and an abnormality in a membrane glycoprotein, we performed similar studies on the giant platelets from two patients with the May-Hegglin anomaly. The patients' platelet electrophoretic mobilities did not differ from control. Although the total sialic acid contents of the patients' platelets were greater than control when calculated per platelet, they were very similar to control when normalized for differences in platelet volume and surface area. When platelet proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis there were no differences between the glycoproteins of control and patient platelets as judged by the patterns of periodic acid Schiff staining and fluorescein-labeled concanavalin A binding. Similarly, patterns of surface glycoprotein labeling by neuraminidase/galactose oxidase/KB3H4 were identical. We conclude that unlike the giant platelets in the Bernard-Soulier syndrome, those of the May-Hegglin anomaly are not associated with a membrane abnormality detectable by these techniques.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

398 Lipid Trafficking in Epithelial Cells: Structure-Function Studies On the Cholera Toxin Receptor Ganglioside GM1

2009 ◽  
Vol 136 (5) ◽  
pp. A-67
Author(s):  
Daniel Chinnapen ◽  
Wendy Hamman ◽  
Jessica Wagner ◽  
M. David Ullman ◽  
Wayne I. Lencer
Keyword(s):  
Epithelial Cells ◽  
Structure Function ◽  
Cholera Toxin ◽  
Ganglioside Gm1 ◽  
Lipid Trafficking ◽  
Toxin Receptor

Discriminating between Cell Surface and Intracellular Plasminogen-binding Proteins: Heterogeneity in Profibrinolytic Plasminogen-binding Proteins on Monocytoid Cells

2000 ◽  
Vol 84 (11) ◽  
pp. 882-890 ◽  
Author(s):  
Michael Green ◽  
Lindsey Miles ◽  
Stephen Hawley
Keyword(s):  
Cell Surface ◽  
Peripheral Blood ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Annexin Ii ◽  
Two Dimensional ◽  
Intact Cells ◽  
Carboxyl Terminal

SummaryWhen plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membraneassociated α-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to α-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated α-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface. The abbreviations used are: 2D, two-dimensional; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; BSA, bovine serum albumin; CpB, carboxypeptidase B; EACA, є-aminocaproic acid; HBSS, Hanks’ Balanced Salt Solution supplemented with 20 mM HEPES; HBSS-BSA, HBSS with 0.1% bovine serum albumin; HRP, horseradish peroxidase; IEF, isoelectric focusing; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBST, Tris buffered saline with 0.1% Tween 20; uPAR, urokinase-type plasminogen activator receptor.

scholarly journals Iodinated fibroblast β-glucuronidase as a ligand for receptor-mediated endocytosis

1985 ◽  
Vol 229 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M F Dean ◽  
S Diment ◽  
C Ostlünd ◽  
B M Jenne ◽  
S Contractor
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peritoneal Macrophages ◽  
Affinity Column ◽  
3T3 Cells ◽  
Receptor Mediated Endocytosis ◽  
3T3 Fibroblasts ◽  
Different Types ◽  
Mannose Receptors ◽  
Mouse Peritoneal Macrophages

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 × 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 × 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

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