Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

Modulation of endothelial cell surface glycoconjugate expression by organ-derived biomatrices

Cell surface molecules play an important role in cellular communication, migration, and adherence. Here, we show the effect of organ-derived biomatrices on endothelial cell surface glycosylation. Five different lectins (with and without neuraminidase treatment) have been used as probes in an enzyme-linked lectin assay to quantitatively detect glycoconjugates on endothelial cells (BAEC) grown on tissue culture plastic or biomatrices isolated from bovine lung, liver, and kidney. BAEC generally exhibit strong binding of concanavalin A (Con A), Ricinus communis agglutinin I (RCA-I), wheat germ agglutinin (WGA), and soybean agglutinin, and peanut agglutinin after neuraminidase pretreatment of cells (Neu-SBA and Neu-PNA), while SBA and PNA consistently bind weakly to BAEC. BAEC grown on organ-derived biomatrices exhibit significantly altered binding intensities of Con A, RCA-I, WGA, and Neu-PNA: BAEC cultured on lung- or kidney-derived biomatrices express significantly stronger binding affinities for Con A and RCA-I than BAEC grown on liver-derived biomatrix or tissue culture plastic. In contrast, BAEC binding of WGA and PNA (after treatment of cells with neuraminidase) is significantly reduced when BAEC are grown on liver- or kidney-derived biomatrix. Quantitative lectin immunogold electron microscopy reveals consistently stronger lectin binding over nuclear regions compared to junctional regions between neighboring cells. These results indicate that extracellular matrix components regulate endothelial cell surface glycoconjugate expression, which determines cellular functions, e.g., preferential adhesion of lymphocytes or metastatic tumor cells.     

Comparison of desialylation of rat transferrin by cellular and non-cellular methods.

We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.     

The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.     

Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [(3)H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A-Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A-Sepharose and these glycoproteins could be eluted with solutions containing methyl alpha-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl alpha-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [(3)H]fucose-labelled glycoproteins from adult myelin and [(14)C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205-212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.     

SOLUBLE AND MEMBRANE LECTIN-BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE

Abstract— Fluorescein isothiocyanate-labelled lectins were used to identify lectin-binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin-binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin-binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin-60, and 7 Ricinus communis agglutinin-120 (RCA-120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A-binding bands and one of the RCA-120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β-hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.     

Lectin-enzyme immunoassay of transferrin sialovariants using immobilized antitransferrin and enzyme-labeled galactose-binding lectin from Ricinus communis

A heterologous lectin-enzyme immunoassay is described. Microtiter plate wells were coated with affinity-purified antibodies to human transferrin. After incubation with transferrin sialovariants, prepared by limited neuraminidase treatment and separated with chromatofocusing, a lectin-enzyme-streptavidin complex was added. A good correlation was obtained between the number of terminal galactose groups on transferrin and the response in the lectin-enzyme immunoassay using Ricinus communis agglutinin as the galactose-binding lectin. The results indicate that characterization of glycosylation is possible with less than a microgram of the glycoprotein available, using lectin-enzyme immunoassays.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation

In this study we report that bone and platelet osteonectin are structurally and functionally heterogeneous in terms of glycosylation and collagen binding capacity. The relative sensitivity of bone and platelet osteonectin to specific glycosidases was used to evaluate potential differences in glycosylation. Although native bone and platelet osteonectin are electrophoretically nonidentical, N-glycanase treatment yielded products with the same apparent molecular weight. Bone osteonectin was also susceptible to cleavage by endo H but not to neuraminidase, while platelet osteonectin was susceptible to neuraminidase but not to endo H. In lectin blotting experiments of bone and platelet osteonectin, concanavalin A bound specifically to bone osteonectin but not to platelet osteonectin. However, Lens culinaris agglutinin bound to platelet osteonectin but not to bone osteonectin. These data suggest that bone and platelet osteonectin differ in their oligosaccharide side chain structures, with bone osteonectin possessing a high mannose-type and platelet osteonectin, a complex-type structure. Solid-phase binding techniques were used to functionally evaluate bone and platelet osteonectin in terms of collagen binding. Although bone osteonectin bound specifically to types I, III, and V collagen, platelet osteonectin had no apparent affinity for these collagen types suggesting that the two proteins are also functionally distinct.     

The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.     

Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)