Affinity chromatographic isolation of cell surface glycoproteins from human foetal brains and their interaction with lectins.

The cell surface glycoproteins of foetal human neurons and glial cells were isolated by affinity chromatography on Con A-Sepharose 4B. Dissociation of Con A from the matrix took place independent of buffer composition and the absence of lipids and/or detergents during chromatography. It was apparently related to the nature of glyco proteins. Pretreatment of Con A-Sepharose 4B with urea or guanidine minimized this problem. The elution of glycoproteins from the affinity matrix at 4 degrees C, instead of the usual 25 degrees C, reduced both Con A and glycolipid contamination in the eluate. Dot-enzyme-linked-lectin assay was carried out with horse radish peroxidase conjugated lectins and serotonin. It was observed that total glycoproteins contained high mannose, hybrid and a limited quantity of biantennary complex type oligosaccharide chains. O-linked oligosaccharides were also present. Desialylation and sodium chloride inhibited binding to serotonin and wheat germ agglutinin indicating the presence of sialic acid residues. Fucose was attached to the innermost core GlcNAc residue, as revealed by affinity towards pea lectin.     

Purification of the glycoprotein allergen Ag7 from mugwort pollen by concanavalin A affinity chromatography.

Two affinity columns comprising immobilized concanavalin A (Con A), Con A-Sepharose and Con A-XP3507, were evaluated for their purifying ability for the glycoprotein allergen Ag7 from a partially purified extract of mugwort pollen. The most pronounced difference between the two columns was the nature of their nonspecific interactions; hydrophobic interactions were dominant with Con A-XP3507, whereas ionic interactions were dominant with Con A-Sepharose. Both Con A-columns were effective for purifying Ag7 with a recovery of 50% after specific elution with displacing sugars. The inclusion of 1.0 M NaCl and 20% ethylene glycol in the elution medium was useful for desorbing nonspecifically bound material, prior to specific elution of adsorbed Ag7 in the presence of the displacing sugars, alpha-methyl glucoside and alpha-methyl mannoside. The most efficient purification of Ag7 was achieved with Con A-Sepharose at room temperature rather than at 4 degrees C. Affinity chromatography with Con A-XP3507 resulted in a slightly more contaminated product (purity 54%) than with Con A-Sepharose (purity 64%).     

Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, JM, Warth, R. and Mu?oz, E. (1978) FEBS Letter, 86, 1-5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (alpha, beta, gamma, delta and epsilon) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Isolation of a concanavalin A receptor from mouse L cells.

A cell surface glycoprotein receptor for concanavalin A (Con A) has been isolated from mouse L cells. The isolation procedure involved dissolving whole L cells in 0.3 M lithium diiodosalicylate and extracting with aqueous phenol. The Con A receptor, which was found in the aqueous phase of this extract, was further purified by affinity chromatography on a column of Con A-Sepharose; the receptor was adsorbed to Con A-Sepharose and eluted with 0.1 M methyl alpha-D-glucopyranoside or with 0.1 M methyl alpha-D-mannopyranoside, but not with other monosaccharides. The cell surface location of the Con A receptor purified in this way was confirmed by showing that it can be isolated from purified L cell plasma membranes and by demonstrating that it can be labeled from the exterior surface of intact L cells by the nonpenetrating galactose oxidase-KB3H4 system. Biochemical studies of the Con A receptor have shown that it migrates on sodium dodecyl sulfate-polyacrylamide gels as a single component having an apparent molecular weight of approximately 100,000. Its N-terminal amino acid is valine and it has carbohydrate attached at several (at least five) different sites along the polypeptide chain.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius

The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Preparation of concanavalin A free purified alpha-1-antitrypsin.

A simple technique is described to remove traces of concanavalin A (Con A) from human α-1-antitrypsin (α-1-AT) purified on commercially available Con A-Sepharose. The α-1-AT was fractionated from serum by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, Con A-Sepharose, and activated thiol-Sepharose at 4°C with solution pH ranges of 7.4–7.6 in all steps. Contaminating Con A was easily removed by binding α-1-AT through the reactive sulfhydryl group to the activated thiol-Sepharose gel and washing away the contaminating Con A with a solution of methyl-α-d-glucopyranoside before final elution of bound α-1-AT. This simple procedure yields purified α-1-AT free of traces of Con A. The α-1-AT was obtained in overall yields of 40–48% from serum with an average molecular weight of 53,500 ± 3000 determined on 15% disc polyacrylamide gels containing sodium dodecyl sulfate (SDS). The isolated α-1-AT exhibited unaltered Pi M phenotype compared to serum α-1-AT but contained traces of several other serum proteins.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.     

Purification of alpha-amylase isoenzymes from Scytalidium thermophilum on a fluidized bed of alginate beads followed by concanavalin A-agarose column chromatography

An alpha-amylase has been purified from the thermophilic fungus Scytalidium thermophilum. A ninefold purification was achieved in a single step using fluidized bed chromatography wherein alginate was used as the affinity matrix. There are at least two isoenzymes as shown by concanavalin A (Con A)-agarose column chromatography. The isoenzyme binding to Con A is stable for at least 3 h at 80 degrees C in the presence of calcium ions. The isoenzymes have similar molecular weights of around 45,000 Da as shown by SDS-PAGE analysis. The isoenzymes differ only slightly in their pH optima and temperature optima but the isoenzyme binding to Con A-agarose has slightly higher thermal stability.     

Properties of the isoenzyme forms A-1, A-2 and B of N-acetyl-beta-D-glucosaminidase purified from baboon kidneys

Three forms of N-acetyl-beta-D-glucosaminidase (NAG: A, B and I) were separated from baboon kidney using Con A-Sepharose and DEAE-Trisacryl chromatography. 2. The A form was further purified into two forms A-1 and A-2 using hydroxylapatite chromatography and anodic PAGE. Both were homogeneous on SDS-PAGE and anodic PAGE but microheterogeneous on PAG-IEF, which could be eliminated by prior treatment with endoglycosidase H or glycopeptidase F. 3. The carbohydrate content accounted for some of this microheterogeneity since it varied from 31 for A-1 to 17% for A-2 and the sialic acid was 6 and 1%. Deamidation may also contribute since the acidic amino acids (29 mol%) and ammonia were high following acid hydrolysis. 4. The mol. wt for A-1, determined by SDS-PAGE, was 52.1 K. 5. The pH optimum was 4.55 and the pI4.97. 6. The optimum temperature for NAG A and B was 50 degrees and 42 degrees C, but B retained more activity above 55 degrees C. 7. The Km for N-acetyl-beta-D-glucosamine and -galactosamine for both isoforms was 0.497 and 0.627 mM respectively. 8. Several ions were found to be uncompetitive inhibitors. Ag+ and Pb2+ were the most potent having Ki values of 3.6 and 8.5 mM respectively. Acetate acted as a competitive inhibitor.     

Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis

A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods. The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method. Phosphorus contents were less than 0.01% of the polysaccharide. Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively. The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan. The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6). A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.     

Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid.

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.     

Purification and properties of a novel latent proteinase showing myosin heavy chain-degrading activity from threadfin-bream muscle

A novel latent proteinase of which activity was induced by heating in the presence of NaCl was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60 degrees C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.     

Mouse ovarian factors with angiogenic activities: partial characterization.

Extracts of nonluteal mouse ovaries (Jcl: ICR strain) were assayed for neovascularization by implanting Elvax films, impregnated with test samples, on the lateral wall of the sheath of m. rectus abdominis in young adult mice of the same strain. Neovascularization occurred in a dose-dependent manner. Angiogenic activity was increased in extracts of ovaries from mice treated with follicle-stimulating hormone (FSH). The angiogenic activity was retained when the extract was heated to 100 degrees C for 15 min or adsorbed with activated charcoal. The ovarian extract was purified by affinity chromatography on a Con A-Sepharose 4B column. The adsorbed fraction possessed higher angiogenic activity. The ovarian extract was fractionated by ammonium sulfate precipitation. High angiogenic activity was found in the fraction collected between 20 to 50% saturation.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.