scholarly journals The influence of degree-of-branching and molecular mass on the interaction between dextran and Concanavalin A in hydrogel preparations intended for insulin release

2012 ◽  
Vol 80 (1) ◽  
pp. 143-148 ◽  
Author(s):  
Ian Benzeval ◽  
Adrian Bowyer ◽  
John Hubble
Keyword(s):  
Molecular Mass ◽  
Insulin Release ◽  
Concanavalin A ◽  
Degree Of Branching

scholarly journals Purification and comparison of the structures of human liver acidic α-d-mannosidases A and B

1986 ◽  
Vol 233 (1) ◽  
pp. 65-72 ◽  
Author(s):  
S H Cheng ◽  
S Malcolm ◽  
S Pemble ◽  
B Winchester
Keyword(s):  
Molecular Mass ◽  
Isoelectric Focusing ◽  
Concanavalin A ◽  
Human Liver ◽  
Large Subunit ◽  
Small Subunit ◽  
Single Locus ◽  
Structure And Properties ◽  
Molecular Masses ◽  
High Mannose

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.

scholarly journals Isolation of a glycoprotein responsible for the enhanced concanavalin A agglutinability of erythrocytes in Yoshida-ascites-sarcoma-bearing rats: the mechanism of paraneoplastic syndromes

1993 ◽  
Vol 292 (1) ◽  
pp. 163-170 ◽  
Author(s):  
R D Kalraiya ◽  
A Sanjay ◽  
N G Mehta
Keyword(s):  
Molecular Mass ◽  
Concanavalin A ◽  
Paraneoplastic Syndromes ◽  
Host Cells ◽  
Ascites Fluid ◽  
Con A ◽  
Tumour Bearing ◽  
Apparent Homogeneity

As a model for the development of paraneoplastic syndromes, we have studied the mechanism by which erythrocytes in the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma acquire higher agglutinability with concanavalin A (Con A). The in vitro incubation of erythrocytes from normal animals with the cell-free ascites fluid or the plasma of tumour-bearing animals is able to confer an enhanced agglutinability on the cells. Fractionation of the ascites fluid has yielded three subfractions that are active in vitro. Two of these, occurring in small amounts, are a particulate fraction rich in plasma-membrane markers and a soluble fraction containing protein of molecular mass equal to or less than 50 kDa. These two are, however, unable to affect the agglutinability of erythrocytes in vivo, i.e. when injected intraperitoneally into normal rats. The third, and major, fraction consists of proteins of molecular mass equal to or greater than 680 kDa, and is able to modify the erythrocyte agglutinability in vivo. From this fraction, by using a combination of Con A affinity chromatography, gel filtration, (NH4)2SO4 fractionation and DEAE-Sephadex chromatography, an active protein has been purified to apparent homogeneity. It yields a subunit of 310 kDa in the presence of SDS and further breaks down into a polypeptide of 170 kDa when reduced with 2-mercaptoethanol. It has a pI of 5.35. The protein is rich in Glx, and appears to contain hybrid-type N-linked oligosaccharides. The protein is also present in the blood plasma of tumour-bearing, but not normal, rats. The radioiodinated protein binds to the erythrocyte surface adding about 7400 molecules/cell. The study unequivocally demonstrates that a protein from the tumour fluid can appear in the circulation, interact with host cells that are not in contact with the tumour and modify their properties.

scholarly journals Effects of Bordetella pertussis toxin on catecholamine inhibition of insulin release from intact and electrically permeabilized rat islets

1989 ◽  
Vol 258 (3) ◽  
pp. 669-675 ◽  
Author(s):  
S J Persaud ◽  
P M Jones ◽  
S L Howell
Keyword(s):  
Molecular Mass ◽  
G Proteins ◽  
Insulin Release ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Pancreatic Beta Cells ◽  
Specific Binding ◽  
Rat Islets ◽  
Bordetella Pertussis Toxin

Noradrenaline- and clonidine-induced inhibition of insulin release from intact and electrically permeabilized rat islets was markedly relieved by prior exposure to 100 ng of Bordetella pertussis toxin/ml. The reversal of catecholamine inhibition of insulin secretion by this toxin was not associated with a decrease in specific binding of the alpha 2-adrenergic ligand [3H]yohimbine, and could not be fully explained by an increase in intracellular cyclic AMP. Exposure of intact islets to 1 microgram of pertussis toxin/ml for 2 h, followed by electrical permeabilization and incubation with 5 microCi of [alpha-32P]NAD+, resulted in the ADP-ribosylation in situ of a protein of molecular mass approx. 41 kDa. These results suggest that pertussis toxin alleviates catecholamine inhibition of beta-cell secretory responses by ADP-ribosylating at least one protein of molecular mass 41 kDa. In analogous systems the 41 kDa substrate of pertussis toxin has been shown to be the alpha subunit of Gi, but catecholamine-activated G proteins linked to effector systems other than adenylate cyclase might also be modified by this toxin in pancreatic beta-cells.

Effect of Salt Stress on the Production and Properties of Extracellular Polysaccharides Produced by Cryptococcus laurentii

2005 ◽  
Vol 60 (5-6) ◽  
pp. 444-450 ◽  
Author(s):  
Emília Breierová ◽  
Zdenka Hromádková ◽  
Eva Stratilová ◽  
Vlasta Sasinková ◽  
Anna Ebringerová
Keyword(s):  
Salt Stress ◽  
Molecular Mass ◽  
Structural Features ◽  
Nacl Stress ◽  
Stress Conditions ◽  
Yeast Cells ◽  
Extracellular Polysaccharides ◽  
Gel Chromatography ◽  
Cryptococcus Laurentii ◽  
Degree Of Branching

The composition, main structural features and molecular properties of exopolysaccharides (EP) produced by Cryptococcus laurentii var. laurentii CCY 17-3-16 under optimal (EPo) and NaCl-stress conditions (EPs) as well as their subfractions isolated by gel chromatography were studied using chemical, FT-IR and NMR spectroscopy methods. The results showed that under stress conditions the yeast produced EP with a lower content of protein and phosphorus. In comparison to EPo, the EPs exhibited a substantially larger proportion of high molecular mass populations. NMR analysis of EPs revealed a higher degree of branching with single xylose side chains of the heteromannan components. The increase of the molecular mass and degree of branching of the macromolecular chains of the heteromannan components might in part be related to the function of EPs to protect the yeast cells from water loss and maintain growth conditions under the salt stress.

scholarly journals Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells

1990 ◽  
Vol 265 (1) ◽  
pp. 121-129 ◽  
Author(s):  
S R Hull ◽  
Z Sheng ◽  
O Vanderpuye ◽  
C David ◽  
K L Carraway
Keyword(s):  
Cell Surface ◽  
Molecular Mass ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Mammary Adenocarcinoma ◽  
Velocity Sedimentation ◽  
Lectin Affinity ◽  
High Mannose ◽  
Sialomucin Complex ◽  
Triton X

Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.

Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

Biologia ◽  
2009 ◽  
Vol 64 (2) ◽  
Author(s):  
Dana Flodrová ◽  
Soňa Garajová ◽  
Anna Malovíková ◽  
Danica Mislovičová ◽  
Jiřina Omelková ◽  
...  
Keyword(s):  
Activation Energy ◽  
Cell Wall ◽  
Molecular Mass ◽  
Isoelectric Point ◽  
Concanavalin A ◽  
Degree Of Polymerization ◽  
Substrate Preference ◽  
Ph Optimum ◽  
Main Form ◽  
Enzyme Substrates

AbstractThe main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others, purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol). The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.

scholarly journals Characterization of a Zn2+-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity

1992 ◽  
Vol 286 (2) ◽  
pp. 435-440 ◽  
Author(s):  
D E Sok ◽  
M R Kim
Keyword(s):  
Bacillus Cereus ◽  
Molecular Mass ◽  
Mouse Brain ◽  
Concanavalin A ◽  
Heat Denaturation ◽  
Phosphodiesterase Activity ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Optimal Ph

p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.

scholarly journals Identification and characterization of the major stilbene-disulphonate- and concanavalin A-binding protein of the porcine renal brush-border membrane as aminopeptidase N

1990 ◽  
Vol 271 (1) ◽  
pp. 147-155 ◽  
Author(s):  
H See ◽  
R A F Reithmeier
Keyword(s):  
Molecular Mass ◽  
Concanavalin A ◽  
Brush Border ◽  
Membrane Fraction ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Aminopeptidase N ◽  
Brush Border Membranes ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

A 130 kDa glycoprotein (GP 130) was purified from porcine renal brush-border membranes by affinity chromatography using immobilized 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate (SITS)- and concanavalin A-Sepharose. GP 130 was the major concanavalin A-binding protein in porcine renal brush-border membranes and also bound Ricinus communis (castor-bean) and wheat-germ agglutinins. Endo-beta-N-acetylglucosaminidase F reduced the molecular mass of GP 130 by 20 kDa as determined by SDS/PAGE, whereas endo-beta-N-acetylglucosaminidase H reduced the molecular mass by 5 kDa, showing that GP 130 contained both complex and high-mannose carbohydrate structures. Western-blot analyses using an antibody raised against GP 130 showed that it was localized to the brush-border membrane fraction and was present in a membrane fraction of the pig kidney cell line LLC-PK1. The N-terminal sequence and amino acid composition of GP 130 showed that GP 130 is similar to rat kidney zinc peptidase and human intestinal aminopeptidase N. GP 130 had aminopeptidase N enzymic activity and was inhibited by bestatin (Ki = 36 microM), 1,10-phenanthroline (Ki 30 microM), Zn2+ (Ki 26 microM), Cu2+ (Ki 260 microM), pre-incubation with EDTA and by a polyclonal antibody against GP 130. Bicarbonate and iodide blocked the binding of GP 130 to the SITS-affinity resin, showing that GP 130 has an anion-binding site. Neither these anions nor stilbene disulphonates affected the aminopeptidase N activity of GP 130.

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