Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

1990 ◽  
Vol 68 (1) ◽  
pp. 199-209 ◽  
Author(s):  
Douglas L. Hogue ◽  
Kevin C. Hodgson ◽  
Carol E. Cass
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nucleoside Transport ◽  
Leukemia Cells ◽  
Relative Mass ◽  
Complex Type ◽  
Trifluoromethane Sulfonic Acid ◽  
L1210 Cells

Membrane polypeptides (relative mass (Mr) 48 000 – 55 000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42 000 – 47 000) during sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS–polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41 000 – 48 000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by > 95% had little, if any, effect on either the affinity (Kd values, 0.1–0.2 nM) or abundance (Bmax values, 200 000 – 220 000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 °C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.Key words: nucleoside transport, L1210 cells, nitrobenzylthioinosine, glycoproteins, tunicamycin.

Chemical and genetic comparison of the glucose and nucleoside transporters

1986 ◽  
Vol 64 (11) ◽  
pp. 1170-1180 ◽  
Author(s):  
Amira Klip ◽  
Denise Walker ◽  
Amos Cohen ◽  
Chungyee Leung
Keyword(s):  
Glucose Transport ◽  
Cytochalasin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Inhibitor ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Relative Mass ◽  
Transport Function ◽  
Sds Page

Glucose and nucleoside uptake into human red cells occurs through protein(s) which copurify in a complex, known as band 4.5 of relative mass (Mr) 66 000 to 50 000. The specific inhibitor of glucose transport, [3H]cytochalasin B, and the specific inhibitor of nucleoside transport, [3H]nitrobenzylthioribofuranosylpurine ([3H]NBMPR), incorporate covalently into component(s) of band 4.5 upon irradiation with ultraviolet light. Both photolabelled components are shown to be glycoproteins, since their migration in sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) is increased after treatment of photolabelled band 4.5 with endoglycosidase F. Peptide maps of the photolabelled components were compared. Red cell membranes were photolabelled with either [3H]cytochalasin B or [3H]NBMPR and subjected to SDS–PAGE. The region containing band 4.5 was cut and transferred to a second SDS–PAGE system and exposed to either papain or Staphylococcus aureus V8 protease. Papain (5 μg) completely cleaved band 4.5 and produced fragments of Mr 33 000, 26 000, 21 000, 15 000, and 12 500. Of these, the 21 000 fragment was the most conspicuous and it retained the label of [3H]cytochalasin B; the 33 000 fragment retained the label of [3H]NBMPR. The V8 protease (0.75 μg) completely cleaved band 4.5 and produced fragments of Mr 35 000, 28 000,22 000, 16 000,13 500, and 9000. The 28 000 fragment retained the label of [3H]cytochalasin B. The label of [3H]NBMPR was distributed along the gel in several regions comprising the 35 000, 28 000, and 16 000 fragments. Longer treatment with the V8 protease did not alter the position of the 28 000 [3H]cytochalasin B labelled peak, but completely abolished the [3H]NBMPR labelled peaks. Genetic segregation of the glucose and nucleoside transporters was determined in a lymphoma cell line. A mutant (14T−g) of S49 cells was selected which had lost the capacity to transport thymidine or to bind NBMPR. Uptake of either 2-deoxyglucose or 3-O-methylglucose, inhibitable by cytochalasin B, was not impaired in this mutant. It is concluded that the nucleoside and glucose transporters are glycoprotein components of band 4.5, which are differentiated by peptide map analysis. Further, a lymphoblast mutant was isolated which had lost the nucleoside transport function but retained the glucose transport function.

scholarly journals Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

scholarly journals Structural characteristics of Tla products.

1985 ◽  
Vol 162 (3) ◽  
pp. 781-789 ◽  
Author(s):  
M J Chorney ◽  
J S Tung ◽  
Y Bushkin ◽  
F W Shen
Keyword(s):  
Peptide Mapping ◽  
Biochemical Study ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromosome 17 ◽  
Leukemia Cells ◽  
Mouse Strains ◽  
Chymotryptic Peptide ◽  
Sds Page

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.

The anaerobic sn-glycerol-3-phosphate dehydrogenase: cloning and expression of the glpA gene of Escherichia coli and identification of the glpA products

1982 ◽  
Vol 60 (3) ◽  
pp. 224-231 ◽  
Author(s):  
Anthony Schryvers ◽  
Joel H. Weiner
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phosphate Transport ◽  
Cloning And Expression ◽  
Relative Mass ◽  
Recombinant Plasmids ◽  
Sds Page ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Definition Of ◽  
Different Strains

The expression of recombinant plasmids carrying the glpA gene (anaerobic glycerol-3-phosphate dehydrogenase) and the closely linked glpT gene (glycerol 3-phosphate transport) were studied under aerobic and anaerobic conditions. When the pattern of expression of enzymatic activity in different strains was compared with sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE) analysis from the same cells the glpA products were identified. Two polypeptides of 62 000 and 43 000 relative mass correlated with enzymatic activity.Five recombinant plasmids that contained one or both of the glpT or glpA genes were isolated and subjected to restriction endonuclease cleavage analysis. The regions of overlap from the inserts in these plasmids allowed definition of the regions of DNA containing the glpT and glpA genes. SDS–PAGE analysis revealed a partial product of the glpA locus from one plasmid, pLC42-17, which allowed more precise definition of the glpA locus on the physical DNA map and prediction of the direction of transcription.

scholarly journals An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4)

1981 ◽  
Vol 200 (1) ◽  
pp. 83-91 ◽  
Author(s):  
I C Madley ◽  
B D Hames
Keyword(s):  
Binding Sites ◽  
Bacterial Contamination ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Radioactivity ◽  
Wild Type ◽  
Stage Of Development ◽  
Affinity Constants ◽  
Radioactivity Measurements

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.

scholarly journals Purified mu EBP-E binds to immunoglobulin enhancers and promoters.

1988 ◽  
Vol 8 (11) ◽  
pp. 4972-4980 ◽  
Author(s):  
C L Peterson ◽  
S Eaton ◽  
K Calame
Keyword(s):  
Heavy Chain ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Apparent Homogeneity ◽  
Enhancer Binding Protein ◽  
Chemical Nuclease

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.

scholarly journals PROTEINS OF THE POSTSYNAPTIC DENSITY

1974 ◽  
Vol 63 (2) ◽  
pp. 456-465 ◽  
Author(s):  
G. Banker ◽  
L. Churchill ◽  
C. W. Cotman
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Synaptic Plasma Membrane ◽  
Brain Protein ◽  
Single Polypeptide ◽  
Structural Matrix ◽  
Polypeptide Band

An analysis was made of the protein composition of a fraction of postsynaptic densities (PSDs) prepared from rat brain. Protein makes up 90% of the material in the PSD fraction. Two major polypeptide fractions are present, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major polypeptide fraction has a molecular weight of 53,000, makes up about 45% of the PSD protein, and comigrates on gels with a major polypeptide of the synaptic plasma membrane. The other polypeptide band has a molecular weight of 97,000, accounts for 17% of the PSD protein, and is not a prominent constituent of other fractions. Six other polypeptides of higher molecular weight (100,000–180,000) are consistently present in small amounts (3–9% each). The PSD fraction contains slightly greater amounts of polar amino acids and proline than the synaptic plasma membrane fraction, but no amino acid is usually prominent. The PSD apparently consists of a structural matrix formed primarily by a single polypeptide or class of polypeptides of 53,000 molecular weight. Small amounts of other specialized proteins are contained within this matrix.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits

1982 ◽  
Vol 60 (4) ◽  
pp. 463-470 ◽  
Author(s):  
T. Youdale ◽  
J. P. MacManus ◽  
J. F. Whitfield
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Purification And Properties ◽  
Exclusion Chromatography

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

scholarly journals Changes in glycan branching and sialylation of the Thy-1 antigen during normal differentiation of mouse T-lymphocytes

1985 ◽  
Vol 226 (2) ◽  
pp. 519-525 ◽  
Author(s):  
S R Carlsson
Keyword(s):  
T Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cell Types ◽  
Complex Type ◽  
Antigen Present ◽  
High Mannose ◽  
Normal Differentiation ◽  
Immature Cells

The glycans of the Thy-1 antigen present on thymocytes and lymph-node T-lymphocytes were investigated after external labelling of the cells. Neuraminidase, endoglycosidase H and endoglycosidase F were used in combination with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing in order to characterize the nature of the glycans on 125I-labelled and immunoprecipitated Thy-1. Glycopeptides were prepared from Thy-1 obtained from cells labelled by periodate/boro[3H]hydride treatment. The glycopeptides were separated by affinity chromatography on concanavalin A-Sepharose and analysed by gel filtration. The results show that both types of cells possess Thy-1 molecules with three N-linked carbohydrate chains, of which one is of ‘high-mannose’ type and the other two of triantennary and biantennary ‘complex’ type. The ratio of triantennary/biantennary chains was decreased on Thy-1 of mature cells compared with that of immature cells, but instead more sialic acid was present on these chains. Deglycosylated Thy-1 appeared to be of the same size regardless of origin, indicating that only the carbohydrate moiety differs between Thy-1 molecules of the two cell types.

scholarly journals The distinguishing characteristics of the plasma membrane are its exposed proteins

1975 ◽  
Vol 147 (2) ◽  
pp. 373-376 ◽  
Author(s):  
R E Gates ◽  
D R Phillips ◽  
M Morrison
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Types ◽  
Distinguishing Characteristic ◽  
Probe System ◽  
Normal Human

The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.

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