Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.Abbreviations DABITC,4-N,N dimethylaminoazobenzene 4-isothiocyanate - PITC phenylisothiocyanate - Cm carboxymethyl - WGA wheat germ agglutinin - TFA trifluoroacetic acid - PGAP pyroglutamylaminopeptidase     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.     

Transmural exocytosis in maize root cap

Summary The maize root cap is a tissue known for its high production of a fucose-rich slime. At the cellular periphery, two kinds of components exist which are indistinguishable: the cell wall barrier and the slime which passes through. Two complementary probes were used, both at the light and the electron microscope level, in order to distinguish the different components. The lectinUlex europaeus agglutinin I was used as a probe targetting the slime and the enzyme cellulose 1,4--D-glucan cellobiohydrolase I was used to probe the cellulose framework. Both probes were used either alone or sequentially for double labeling. The cytochemical PATAg test was optionally used with the enzyme-gold complex labeling. After several technical improvements (multistep method, increase in accessibility), UeA I was used to follow the exocytic pathway of the slime from the Golgi apparatus to the exterior of the cell. The results indicate the occurrence of at least two populations of Golgi apparatus vesicles, and one is directly engaged in the transport of the fucoserich slime. The slime accumulated in pockets between the plasmamembrane and the outer tangential cell wall. The CBH I-gold complex showed the existence and the maintenance of a thin but continuous cellulosic layer, even when the cells slough. The double labeling showed the fucose-rich compounds within the cell wall. Data emphasize the role of the cell wall as a filtering barrier and a mechanical protection in the course of differentiation.Abbreviations CBH I EC 3.2.1.91, cellulose 1,4--D-glucan cellobiohydrolase I - CMC carboxymethyl cellulose - CPB citrate phosphate buffer - FITC fluorescein isothiocyanate - IC internal cell - PA-TAg periodic acid-thiocarbohydrazide-silver proteinate - PBS phosphate buffered saline - PC cell with accumulation pockets - PEG polyethyleneglycol - SC sloughed cell - UeA I Ulex europaeus agglutinin I - VI UeA I-labelled Golgi-derived vesicles - V2 UeA I-unlabelled Golgi-derived vesicles     

半夏居群遗传分化的同工酶和可溶性蛋白电泳的聚类分析

用聚丙烯酰胺凝胶电泳（PAGE）技术分析了安徽产7个半夏（PinelliaternataBreit）居群的4种同工酶和可溶性蛋白谱带。根据其中的PER、EST和可溶性蛋白谱带的分布特征,用Forguson和Aquado＆Avise的公式分别计算其谱带的相似性系数和遗传距离,以类平均法（UPGMA）进行了聚类分析。结果表明：半夏居群间表现出一定的遗传分化;鹞落坪半夏与半夏的黄山、九华山、大别山万佛寺居群间的遗传距离较小（D≤0.214）,结合形态变异,笔者认为鹞落坪半夏只能是半夏居群中形态上较为特殊的地方宗,而不是一新种;狭叶半夏与其它6个居群的遗传距离较大（D=0.416）,是半夏居群中种系分化较为强烈的一个居群。文中还讨论了半夏的遗传分化与形态变异、生境等的相关性。     

Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity

Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato extensin peroxidase and H₂O₂. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immuno-sorbent assay of the B isoform, using tomato extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato extensin monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457–469). The amino acid and carbohydrate compositions were similar to those of tomato extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possesses an extended polyproline II helix conformation with no evidence of α- helix or β- sheet secondary structure. In conclusion, we refer to this HRGP as potato extensin. As proposed for other extensins, potato extensin is likely to play a role in cell wall architecture and plant disease resistance. Received: 25 November 1996 / Accepted 13 January 1997     

Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

Lectin Analysis of Trypanosoma congolense Bloodstream Trypomastigote and Culture Procyclic Surface Saccharides by Agglutination and Electron Microscopic Technics*

SYNOPSIS. Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark-field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin. Electron-microscopic localization of lectin binding sites on the surface of intact and dextranase-treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of blood-stream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA-, SBA, PP- or FBP-HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions. Intact living bloodstream forms were agglutinated in a concentration-dependent manner with all the lectins tested. Agglutination levels were scored as Con A > FBP > WGA = PP = SBA. Sugars resembling α-D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and α-L-fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin-induced agglutinations were obtained with trypsin- or dextranase-treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A-, SBA- and FBP-binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase-treated cells. No lectin-binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin- or dextranasetreated, procyclics were agglutinated in a concentration-dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A- and WGA-specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin-negative. Thus, T. congolense procyclics retained surface saccharides resembling α-D-mannose and N-acetyl-D-glucosamine but lost sugars resembling N-acetyl-D-galactosamine (or D-galactose) and α-L-fucose. The failure of dextranase to remove the lectin-binding saccharides from the surface of bloodstream and procyclic forms suggests that α-1,6-glucan bonds do not link these carbohydrates. The results are contrasted with lectin research on other trypanosome species and discussed with relation to the biology of T. congolense.     

Lectin-Induced Inhibition of Nerve Growth Factor Binding by Receptors of Sympathetic Ganglia

Abstract: Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on ¹²⁵I-NGF specific binding to preparations of particulate and nonionic detergent-extracted micro-somal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus I, induced a concentration-related, carbohydrate-specific decrease in ¹²⁵T-NGF binding. Following Con A exposure, ¹²⁵I-NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin-associated impairment of NGF binding was also demonstrated by using a Triton X-100 solubilized receptor preparation. These results provide evidence that the high-affinity-state NGF receptor of SCG is a glycoprotein containing N -acetylglucosamine and α-D-mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.