scholarly journals The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

1977 ◽  
Vol 166 (2) ◽  
pp. 217-223 ◽  
Author(s):  
James E. Hudson ◽  
Terry C. Johnson
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
Complete Medium ◽  
Intact Cells ◽  
Membrane Components ◽  
Turn Over ◽  
Glycoprotein Degradation ◽  
Dual Label

When monolayer cultures of neuroblastoma N2a cells were prelabelled with [3H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [14C]fucose and [3H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [3H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents.

scholarly journals Surface redistribution and release of antibody-induced caps in entamoebae.

1980 ◽  
Vol 151 (1) ◽  
pp. 184-193 ◽  
Author(s):  
J Calderón ◽  
M de Lourdes Muñoz ◽  
H M Acosta
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Segregation ◽  
Surface Membrane ◽  
Single Layer ◽  
Surface Antigens ◽  
Spontaneous Release ◽  
Plasma Membrane Regeneration ◽  
Membrane Components ◽  
Radiolabeled Antibodies

Polyspecific antibodies bound to Entamoeba induced surface redistribution of membrane components toward the uroid region. Capping of surface antigens was obtained with a single layer of antibodies in E. histolytica and E. invadens. This surface segregation progressed to a large accumulation of folded plasma membrane that extruded as a defined vesicular cap. A spontaneous release of the cap at the end of the capping process took place. These released caps contained most of the antibodies that originally bound to the whole cell surface. Two-thirds of radiolabeled antibodies bound to the surface of E. histolytica were released into the medium in 2 h. Successive capping induced by repeated exposure of E. invadens to antibodies produced conglomerates of folded surface membrane, visualized as stacked caps, in proportion to the number of antibody exposures. These results indicate the remarkable ability of Entamoeba to rapidly regenerate substantial amounts of plasma membbrane. The properties of surface redistribution, liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.

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 Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.

1975 ◽  
Vol 64 (3) ◽  
pp. 538-550 ◽  
Author(s):  
P P Silva ◽  
A Martínez-Palomo ◽  
A Gonzalez-Robles
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Membrane Structure ◽  
Ruthenium Red ◽  
Iron Binding ◽  
Con A ◽  
Colloidal Iron ◽  
Acidic Sites ◽  
Membrane Components

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.

scholarly journals Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

1985 ◽  
Vol 100 (5) ◽  
pp. 1499-1507 ◽  
Author(s):  
D R Critchley ◽  
P G Nelson ◽  
W H Habig ◽  
P H Fishman
Keyword(s):  
Cell Surface ◽  
Tetanus Toxin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Primary Cultures ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Neuronal Cell ◽  
Subcellular Compartment ◽  
Membrane Components

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

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 Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium

1989 ◽  
Vol 259 (3) ◽  
pp. 645-650 ◽  
Author(s):  
C W Taylor ◽  
M J Berridge ◽  
A M Cooke ◽  
B V L Potter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Racemic Mixture ◽  
Synthetic Analogue ◽  
Intact Cells ◽  
Surface Receptors ◽  
Prolonged Stimulation ◽  
Crude Preparation ◽  
Intracellular Messenger

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.

Role of cell-surface carbohydrates and plasma membrane components in the internalization of cell-penetrating peptides

2010 ◽  
Vol 15 (23-24) ◽  
pp. 1101-1101
Author(s):  
Chérine Bechara ◽  
Chen-Yu Jiao ◽  
Fabienne Burlina ◽  
Isabel D. Alves ◽  
Gérard Chassaing ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating ◽  
Membrane Components ◽  
Surface Carbohydrates

scholarly journals Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules

2018 ◽  
Vol 115 (40) ◽  
pp. E9353-E9361 ◽  
Author(s):  
F. Tudor Ilca ◽  
Andreas Neerincx ◽  
Mark R. Wills ◽  
Maike de la Roche ◽  
Louise H. Boyle
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Receptor ◽  
Target Cells ◽  
Intact Cells ◽  
Mhc I ◽  
Immunogenic Peptides ◽  
Presentation Pathway ◽  
Peptide Exchange

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.

scholarly journals Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

1991 ◽  
Vol 112 (1) ◽  
pp. 39-54 ◽  
Author(s):  
S G Miller ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Secretory Granules ◽  
Intact Cells ◽  
Transport Reaction ◽  
Permeabilized Cells ◽  
Transmembrane Glycoprotein ◽  
Transport Vesicles ◽  
Constitutive Secretion

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

scholarly journals Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase

1988 ◽  
Vol 254 (3) ◽  
pp. 765-771 ◽  
Author(s):  
D Allan ◽  
P Quinn
Keyword(s):  
Staphylococcus Aureus ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Fluid Phase ◽  
Intact Cells ◽  
Prolonged Incubation ◽  
Bhk Cells ◽  
Streptolysin O ◽  
Incubation Experiments ◽  
Original Cell

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.

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