scholarly journals Isolation and partial characterization of two antigenic glycoproteins from rye-grass (Lolium perenne) pollen

1981 ◽  
Vol 197 (3) ◽  
pp. 695-706 ◽  
Author(s):  
B J Howlett ◽  
A E Clarke
Keyword(s):  
Lolium Perenne ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Lectin Binding ◽  
Sodium Dodecyl ◽  
Binding Assays ◽  
Pea Lectin ◽  
Rye Grass ◽  
Allergenic Activity

Two glycoproteins have been purified from a buffer extract of rye-grass (Lolium perenne) pollen. Both migrated as single bands on sodium dodecyl sulphate/polyacrylamide gels. Glycoprotein 1 (0.8 mg/g of pollen) had a apparent mol.wt. of 33 000 and contained 95% protein and 5% carbohydrate. The monosaccharides glucose, galactose, mannose, arabinose and N-acetylglucosamine were present in the proportions 3:3:1:2:1. Glycoprotein 2 (0.4 mg/g of pollen) had an apparent mol. wt. of 68000 and contained 88% protein and 12% carbohydrate. The monosaccharides glucose, galactose, mannose, fucose, xylose, arabinose and N-acetylglucosamine were present in the proportions 4:7:13:5:8:6:6. This glycoprotein bound concanavalin A and Lotus tetragonolobus (asparagus pea) lectin. Radioallergosorbent (RAST) inhibition tests showed that Glycoprotein 1 is an effective allergen, whereas Glycoprotein 2 has less allergenic activity. A method for performing both lectin-binding assays and RAST inhibition tests using microtitre trays is described.

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

1979 ◽  
Vol 183 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Richard H. Quarles ◽  
Laurence J. McIntyre ◽  
Carol F. Pasnak
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Preceding Paper ◽  
The Other ◽  
Double Labelling ◽  
Myelin Associated Glycoprotein

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. [3H]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 α-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 α-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 [3H]fucose-labelled glycoproteins from adult myelin and [14C]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.

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

scholarly journals A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane

1976 ◽  
Vol 153 (2) ◽  
pp. 265-270 ◽  
Author(s):  
M J A Tanner ◽  
D J Anstee
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Erythrocyte Membrane ◽  
Individual Pattern ◽  
Direct Demonstration ◽  
Staining Techniques ◽  
Membrane Components

1. A method which allows the characterization of lectin-binding components is described. This method should be useful in defining the nature and heterogeneity of these components in cell membranes. 2. The method, which we have used on erythrocyte “ghosts”, involves the fixation of “ghost” components after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and incubation with purified 125I-labelled lectins. 3. Each of the four lectins used shows an individual pattern of reactivity towards “ghosts” components. Band 3, the major membrane-penetrating glycoprotein, is bound by the lectins from Ricinus communis and Phaseolus vulgaris (phytohaemagglutinin) and by concanavalin A. The major erythrocyte sialoglycoprotein is bound by the lectins from R. communis, P. vulgaris and Maclura aurantiaca. 4. Three of the lectins displays binding for other membrane components, some of which are not demonstratable by conventional protein- and carbohydrate-staining techniques.

scholarly journals Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Irene Axarli ◽  
Ariadne Prigipaki ◽  
Nikolaos E. Labrou
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Reaction Rate ◽  
Sodium Dodecyl ◽  
Cytochrome P450s ◽  
Ph Values ◽  
Maximum Value ◽  
Endogenous Compounds ◽  
Ph Range ◽  
Bell Shaped Curve

Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9–8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two ps in the acidic and basic pH range. The results are discussed in relation to the future biotechnology applications of CYPs.

scholarly journals The identification and characterization of two separate carboxylesterases in guinea-pig serum

1983 ◽  
Vol 215 (1) ◽  
pp. 91-99 ◽  
Author(s):  
K Cain ◽  
E Reiner ◽  
D G Williams
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Selective Inhibitor ◽  
Nitrophenyl Phosphate ◽  
Phosphate Treatment ◽  
Pig Serum

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Lectin-binding proteins in central-nervous-system myelin. Detection of glycoproteins of purified myelin on polyacrylamide gels by [3h]concanavalin A binding

1979 ◽  
Vol 183 (2) ◽  
pp. 205-212 ◽  
Author(s):  
L J McIntyre ◽  
R H Quarles ◽  
R O Brady
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
Concanavalin A ◽  
Binding Proteins ◽  
Lectin Binding ◽  
Sodium Dodecyl ◽  
Myelin Proteins ◽  
Polyacrylamide Gels ◽  
Myelin Associated Glycoprotein

Concanavalin A strongly agglutinates purified fragments of immature and mature rat brain myelin, but only weakly agglutinates mature bovine and human myelin fragments. A sensitive method involving [3H]concanavalin binding to sodium dodecyl sulphate/polyacrylamide gels was used to detect the concanavalin A-binding proteins in purified myelin. When applied to mature rat brain myelin proteins that had been labelled in vivo with [14C]fucose, the distribution of the [3H]concanavalin A on the gel was very similar to that of [14C]fucose with the major peak corresponding to the major myelin-associated glycoprotein. The technique revealed that the immature form of the myelin-associated glycoprotein with a slightly larger apparent molecular weight also bound concanavalin A, and that in purified immature rat myelin the quantitative importance of some of the other glycoproteins in binding concanavalin A was increased relative to the myelin-associated glycoprotein. The separated proteins of bovine and human myelin bound more [3H]-concanavalin A than those of rat myelin. In these species, the myelin-associated glycoprotein was a major concanavalin A-binding protein, although two higher-molecular-weight glycoproteins also bound significant quantities of [3H]concanavalin A. The results indicate that there are receptors for concanavalin A on the surface of rat, bovine and human myelin membranes and suggest that the myelin-associated glycoprotein is one of the principal receptors.

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