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.     

Dopamine β-Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis

Abstract: Chromogranin A and two other proteins (A₁ and A₂) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.     

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.     

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)     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.     

BOVINE ADRENAL MEDULLARY DOPAMINE-β-HYDROXYLASE: STUDIES ON INTERACTION WITH CONCANAVALIN A

Bovine adrenal medullary dopamine-β-hydroxylase binds with concanavalin A and forms an enzymically active precipitate. The formation of the insoluble complex is pH-dependent and can be inhibited by α-methyl-D-mannoside, D-mannose and D-glucose. The insoluble complex can be dissociated into two species with α-methyl-D-mannoside. From the results, it appears that the interaction between dopamine-β-hydroxylase and concanavalin A is due to the carbohydrate moiety of dopamine-β-hydroxylase. This property was used to purify the enzyme from a soluble lysate of chromaffin granules. Of all the proteins contained in the soluble lysate, dopamine-β-hydroxylase was the only one to be retained on a column of concanavalin A covalently bound to Sepharose 4B. The preparation of pure dopamine-β-hydroxylase exhibits a very high specific activity of 320 μmol of octopamine formed per 30 min per mg of protein.     

Stimulation of the membrane-bound, magnesium-dependent adenosine triphosphatase of mouse neuroblastoma by concanavalin A and wheat germ agglutinin.

The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg⁺²-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg⁺²-dependent ATPase activity.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.     

Lectin Receptors in Central Nervous System Myelin

Abstract: Proteins from central nervous system myelin were separated by high-resolution, sodium dodecyl sulfate-pore gradient slab gel electrophoresis and the glycoproteins were detected by autoradiography after direct application of radioiodinated lectins. A surprising heterogeneity of lectin binding proteins was found associated with this highly purified membrane fraction. Iodinated wheat germ agglutinin, which has a monosaccharide specificity for N-acetyl-D-glucosamine and N-acetylneuraminic acid, revealed six major bands and two minor bands. By correlating the molecular weights (M_r) of radioiodinated protein standards with the gel concentration at the position reached by the protein (%T) using the relationship log(M_r) versus log(%T) for gradient gel systems, molecular weight estimates of 128, 300, 109, 800, 75, 300, 48, 800, 26, 100 and 23, 700 were obtained for the major glycoprotein bands and molecular weights of 98, 300 and 86, 600 for the minor bands. When the isolated myelin was extracted with chloroform-methanol-a procedure that removes the major myelin proteins, including the proteolipid protein and most of the basic proteins and hence concentrates the minor high molecular weight proteins-and analyzed after gradient gel electrophoresis, additional glycoproteins of molecular weights 607, 700, 196, 900, 175, 100, 61, 800, 52, 200 and 42, 600 were resolved with this lectin. Radioiodinated soybean agglutinin, which has a specificity for N-acetyl-D-galactosamine and D-galactose, revealed seven bands, three of which were unique to this lectin (19, 600, 19, 100 and 17,000). Iodinated concanavalin A (d -mannose, d -glucose) revealed bands similar to the wheat germ agglutinin as well as additional bands of 40, 300, 37, 300, 35, 700, 21, 800 and 20, 400. The glycoprotein specificity for these lectin binding components was demonstrated by hapten carbohydrate binding inhibition and by organic solvent extraction for removal of glycolipids. Based on these experiments using three lectins with different carbohydrate specificity, 22 lectin-reactive components were identified; however, six of these bands were removed by chloroform-methanol extraction. The variations observed in the lectin binding capacity for these different bands suggest possible carbohydrate heterogeneity for these individual glycoproteins. Although many of these bands may be dissociated subunits (monomeric polypeptides) of oligomeric complexes, the observed multiplicity of these quantitatively minor glycoproteins associated with the purified myelin membrane implies a more intricate molecular organization for the myelin sheath complex than previously believed.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.     

The chromaffin granule — Plasma membrane interaction as a model for exocytosis: Quantitative release of the soluble granular content

Bovine adrenal medullary plasma membranes induce the release of soluble components from chromaffin granules in a Ca²⁺ dependent manner. This interaction can be modulated by changing the temperature. Correlation of the concentrations of four released soluble markers (catecholamines, soluble protein, ATP and dopamine-β-hydroxylase) in samples incubated at different temperatures revealed that those markers were liberated simultaneously. Their ratio did not differ significantly from the ratio measured in chromaffin granule lysates. These results suggest the release of the total granular content upon incubation with plasma membranes. Further analysis of the free catecholamines showed a preferential release of adrenalin.     

An application of ellipsometry: Assessment of polysaccharide and glycoprotein interaction with lectin at a liquid/solid interface

Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysacchride, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 and 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 · 10⁷ M^?1 for concanavalin A, 2 · 10⁶ M^?1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl α-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger β-D-galactosidase interacted to different degrees with these lectins. The relative affinity of wheat germ agglutinin for $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ and for chitin-derived oligosaccharides was also determined. When films of sialoglyproteins were treated with neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, usingg little material.     

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-beta-N-acetylglucosaminidase H

A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β-N-acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [¹²⁵I]concanavalin A or [¹²⁵I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [¹²⁵I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [¹²⁵I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N- and O-glycosidic linkages where the oligosaccharide is predominantly mannosyl.     

Dopamine-β-hydroxylase-Induced Changes in the Electrical Conductance of Bimolecular Lipid Membranes

Dopamine-β-hydroxylase (DBH), an enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in adrenal medullary chromaffin granules, increases the electrical conductance of bimolecular lipid membranes. The conductance increase requires both DA and Ca²⁺ and occurs in discrete steps. The conductance, which increases as the square of the DBH concentration, is nonselective for cations over anions and requires the native conformation of DBH. NE cannot replace DA.     

Carbohydrate Composition of Variant-Specific Surface Antigen Glycoproteins from Trypanosoma brucei

SYNOPSIS. The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetyl-glucosamine). The glycoprotein from variant 048, strain 427 contained (±20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an integral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin bands with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120,000).