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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human neuroblastoma SK-N-SH-SY5Y (SY5Y) and rat pheochromocytoma PC12 cells are model cell lines used in the study of nerve growth factor (NGF) effect. The effects of NGF are initiated by binding to cell surface receptors (NGFR). The amino acid sequence for NGFR has been deduced based on the identification of a single gene for NGFR. However, there are two kinds of NGF binding activities and several reported molecular weights of NGFR. We report here on the demonstration of NGFR-like proteins from PC12 and SY5Y cells by sequential lectin chromatography, reverse-phase HPLC, and SDS-PAGE analysis of immunoprecipitates obtained with NGFR-specific monoclonal antibodies. For both human and rodent NGFR, there was a tendency for the higher molecular-weight species of NGFR-like proteins to be eluted in more hydrophobic fractions. Also, the expression of different species of NGFR could be modified by treatment with retinoic acid (RA). These results are consistent with the hypothesis that the different molecular species of NGFR may result from the generation of a truncated form of NGFR, the presence of sugar residues on the NGFR protein, dimer formation between NGFR, or the association of NGFR with a receptor-associated protein.
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PMID:Reverse-phase high-performance liquid chromatography of nerve growth factor receptor-like proteins identified with monoclonal antibodies. 196 79

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).
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PMID:Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis. 198 Nov 41

Our previous work has shown that retinoic acid (RA) enhances fibroblast cell attachment to plastic and to laminin. The treatment of NIH-3T3 cells with RA for 2 days also caused a reproducible increase in the binding of the lectin Phaseolus vulgaris leukoagglutinin (PHA-L) to a glycoprotein of molecular weight 130,000 (gp130) as judged by SDS-PAGE analysis. This finding is consistent with an increased number of beta-1,6-linked N-acetylglucosaminyl residues on gp130. Of the 11 additional lectins tested Ricinus communis agglutinin I (RCA), Phaseolus vulgaris erythroagglutinin (PHA-E), soybean agglutinin (SBA), and succinylated wheat germ agglutinin (sWGA) showed a significant increase in binding specifically to gp130. Similar to RA, 13-cis-RA and 3,5-di-tert-butyl-4-chalcone carboxylic acid, a synthetic retinoid, also increased PHA-L binding to gp130; they also enhanced cell adhesiveness and inhibited cell growth. N-(4-Hydroxyphenyl)-all-trans-retinamide and thyroxine failed to influence adhesion and did not increase PHA-L binding to gp130. Moreover these compounds also failed to inhibit cell growth and to alter the morphology of the cultured cells. Since trypsin is utilized to remove cells from the culture dishes before they are used in the attachment assay to laminin, we studied the effect of this trypsinization step on PHA-L binding to gp130. Trypsin reduced PHA-L binding thus suggesting cell surface localization of gp130. After trypsin treatment RA-treated cells still showed enhanced PHA-L binding compared to dimethyl sulfoxide (DMSO) control. In conclusion RA-induced cell adhesiveness and growth inhibition are accompanied by an increase in the PHA-L, PHA-E, SBA, RCA, and sWGA binding to gp130. The sensitivity of gp130 to trypsin suggests that it is a cell surface glycoprotein.
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PMID:Retinoids enhance lectin binding to gp130, a glycoprotein of NIH-3T3 cells: correlation with cell growth and adhesion. 198 85

Fluorescent lectin binding to cell surfaces was quantitatively analysed by flow cytometry on mortal human breast epithelial cells MCF-10M, the immortalized cell line MCF-10A derived from MCF-10M and sublines of MCF-10A transfected with the neomycin resistance gene (MCF-10Aneo), the c-Ha-ras protooncogene (MCF-10AneoN), or transfected and transformed with the c-Ha-ras activated oncogene (MCF-10AneoT). Immortal MCF-10A cells bound 10-fold more peanut agglutinin (PNA) and soy bean agglutinin (SBA) than did MCF-10M cells. Transformed MCF-10AneoT cells bound approximately ten times more PNA than did non-transformed cells transfected with protooncogene (MCF-10AneoN). Treatment of the transfectants with neuraminidase abrogated the differences in PNA-binding and reduced the differences in SBA binding. SDS-PAGE separation of PNA binding glycoproteins revealed different patterns for all MCF-10A derived sublines.
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PMID:Cell surface glycosylation changes accompanying immortalization and transformation of normal human mammary epithelial cells. 202 77

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules. 204 18

Opsonizing and agglutinating activities of plasma from the freshwater clam, Corbicula fluminea, were found to be inhibited by the sugars, 2-deoxy-D-glucose (deoxy-Glu) and N-acetyl-D-galactosamine (GalNAc). The plasma opsonin/agglutinin was subsequently isolated by a two-step separation procedure. Aldehyde-fixed rabbit erythrocytes (RRBC) were used as a solid-phase plasma opsonin affinity absorbant, and deoxyGlu and GalNAc were used in the eluting buffer to desorb several RRBC-binding plasma proteins. The second step involved the further separation of sugar-eluted proteins by Sephacryl S-200 gel filtration. A plasma protein with an apparent molecular weight of 40 kd on SDS-PAGE under nonreducing conditions was found to possess both agglutinating and opsonizing activities. It was further shown to be composed of two identical 20 kd subunits associated through disulfide linkage(s). Although this protein shares some structural similarity with other bivalve opsonins, differences in native molecular size or subunit structure, agglutinating properties and/or sugar binding specificity support the current hypothesis that naturally occurring plasma opsonins of molluscs represent a heterogeneous group of proteins unified primarily through their lectin-like characteristic of binding specific carbohydrate determinants.
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PMID:Immunorecognition in the freshwater bivalve, Corbicula fluminea. II. Isolation and characterization of a plasma opsonin with hemagglutinating activity. 208 70

The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.
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PMID:A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD. 211 53

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.
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PMID:Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains. 211 44

Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-D-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its beta 1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-beta-D-glucosaminidase, supporting the conclusion that WGA is reacting with terminal beta-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and of glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-D-galactosamine (GalNAc), fucose (Fuc), galactose (Gal), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories.
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PMID:N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin. 212 47

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.
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PMID:Purification to homogeneity and properties of mannosidase II from mung bean seedlings. 213 44


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