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.

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 Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

1984 ◽  
Vol 159 (6) ◽  
pp. 1653-1668 ◽  
Author(s):  
J D Vassalli ◽  
J M Dayer ◽  
A Wohlwend ◽  
D Belin
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Specific Inhibitor ◽  
Mononuclear Phagocytes ◽  
Human Monocytes ◽  
Ligand Complex ◽  
Sds Page ◽  
Protease Nexin

The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

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.

An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts

1984 ◽  
Vol 62 (6) ◽  
pp. 470-478 ◽  
Author(s):  
Safia Wasi ◽  
Kichibee Otsuka ◽  
Kam-Ling Yao ◽  
Pierre S. Tung ◽  
Jane E. Aubin ◽  
...  
Keyword(s):  
Periodontal Ligament ◽  
Culture Medium ◽  
Alveolar Bone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Mass ◽  
Bovine Bone ◽  
Binding Studies ◽  
Periodontal Ligament Fibroblasts ◽  
Sds Page

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine–HCl (GuHCl) and 4 M GuHCl − 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS–PAGE with a relative mass ~ 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented ~0.5% of the total labelled proteins secreted. It comigrated on SDS–PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.

Cytochalasin-B-binding proteins related to glucose transport across the basolateral membrane of the intestinal epithelial cell

1986 ◽  
Vol 85 (1) ◽  
pp. 177-185
Author(s):  
T. Uezato ◽  
M. Fujita
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Basolateral Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Intestinal Epithelial ◽  
Similar Extent ◽  
Basolateral Membranes

Basolateral membrane vesicles were isolated from mouse enterocytes. The vesicles showed Na+-independent uptake of D-glucose. The uptake was inhibited by cytochalasin B and phloretin with 50% inhibition at 2.0 microM and 25 microM, respectively. 2-Deoxy-D-glucose inhibited D-glucose transport by 50% at 0.5 M. The basolateral membranes bound 13.5 pmol mg protein-1 of 0.1 microM-cytochalasin B. The effects of various monosaccharides on cytochalasin B binding were examined; the strongest inhibitor was 2-deoxy-D-glucose (20–30%). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the basolateral membranes labelled with [3H]cytochalasin B revealed two components with Mr values of 52(+/− 2) and 30(X 10(3)). Phloretin and 2-deoxy-D-glucose inhibited the photo-incorporation of [3H]cytochalasin B into these components. While phloretin inhibited the photolabelling of the two components to a similar extent, 2-deoxy-D-glucose seemed to inhibit preferentially that into the 52 X 10(3) Mr component. The similar sensitivity to 2-deoxy-D-glucose of the photolabelling of the 52 X 10(3) Mr component and of D-glucose transport, together with the fact that dithiothreitol removal increased the incorporation into the 52 X 10(3)Mr component and decreased that into the 30 X 10(3) Mr component, seems to suggest that the 52 X 10(3) Mr component is the major glucose transporter of the basolateral membrane.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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