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N Sharon

Publications and source records attributed to N Sharon.

At least 199 records · Page 11Linked to original sources
PubMed1977

Lysozyme.

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Acetylglucosamine↗
PubMed1977

Lectins.

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Binding Sites↗
PubMed1977

Qualitative and quantitative aspects of lectin-induced vacuole formation in mouse peritoneal macrophages.

The binding, internalization, and vacuologenic activity of several native and chemically modified lectins was investigated on untreated and neuraminidase-treated mouse peritoneal macrophages. The distribution of lectin receptors on the cell surface and their internalization was assessed by employing both radioactive and fluorescent lectin derivatives. On the basis of their effect on the macrophages, the lectins tested can be divided into 2 categories: lectins that induce vacuole formation (concanavalin A, wax bean agglutinin, and wheat germ agglutinin), and lectins that do not induce vocuolation (soybean agglutinin, peanut agglutinin, and Lotus tetragonolbus agglutinin). Soybean and peanut agglutinins bound to macrophages only after neuraminidase treatment, but the latter treatment did not change the effect of other lectins on the cells. Glutaraldehyde-cross-linked polymers of soybean or peanut agglutinins, which are multivalent with respect to the number of sugar binding sites, induced vacuolation in neuraminidase-treated cells. On the other hand, succinylation of concanavalin A, which reduces the lectin's valence from 4 to 2 abolished its vacuologenic activity. While the data do not indicate direct correlation between vacuole induction and the number of lectin receptors or the extent of their internalization, they do suggest that multivalency of lectins is an important factor in vacuole formation. Multivalency of a lectin enables extensive cross-linkage of membrane receptors which may be a prerequisite for triggering vacuolation.

Concanavalin A↗
PubMed1976

Chemical modification of the tryptophan residues of wheat-germ agglutinin. Effect on fluorescence and saccharide-binding properties.

The oxidation of the tryptophan residues of wheat germ agglutinin by N-bromosuccinimide was investigated under non-denaturing and denaturing conditions. All three tryptophan residues present in wheat germ agglutinin subunit (molecular weight 18 000) could be modified in 0.1 M acetic acid/8 M urea, pH 3.9. One of the residues failed, however, to react with N-bromosuccinimide when the modification was in 0.1 M citrate buffer, pH 6.0. Tryptophan fluorescence of the protein was quenched concomitantly with the oxidation of two tryptophan residues even when the modification was carried out in acetic acid urea. After oxidation of two tryptophan residues per subunit of wheat germ agglutinin, only 15% of the original tryptophan fluorescence remained; upon excitation at 280 nm, tyrosine fluorescence centered at 305 nm could be resolved. The results suggest that there are only two emitters in the protein and that the third tryptophan residue is buried in the native protein and can be modified only in acetic acid urea. This tryptophan residue is quenched in the native protein. Saturation of wheat germ agglutinin with tri-N-acetylchitotriose did not protect the tryptophan residues from oxidation by N-bromosuccinimide. Under these conditions, however, the reactivity of the tryptophan residues towards N-bromosuccinimide was reduced and a higher concentration of the reagent was required to achieve the same extent of oxidation as in the absence of the saccharide. Oxidation of one tryptophan residue per subunit in acetic acid urea led to almost complete loss (97%) of hemagglutinating activity, a 3.5-fold decrease in the affinity constant for tri-N-acetylchitotriose and loss of ability of the subunits (SO20,w = 2.0 S) to reassociate to the native dimer (So20,w = 3.5 S) after dialysis against a non-denaturing buffer. No significant changes in the circular dichroism spectrum of wheat germ agglutinin were observed after oxidation of the three tryptophan residues, suggesting that no gross conformational changes occurred. The steric relationships between the fluorescent tryptophan residues of wheat germ agglutinin and saccharides are discussed.

Binding Sites↗
PubMed1976

A transition state analog of lysozyme catalysis prepared from the bacterial cell wall tetrasaccharide.

Treatment of the cell wall tetrasaccharide GlcNAcbeta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc with alkali resulted in the formation of the unsaturated tetrasaccharide GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen. The same compound was also formed by transglycosylation upon incubation of the unmodified tetrasaccharide with the unsaturated disaccharide GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen (Tipper, D. J. (1968) Biochemistry 7, 1441-1449) and hen egg white lysozyme. The unsaturated tetrasaccharide was further characterized by paper electrophoresis, amino sugar analysis, and NMR. From NMR analysis it is concluded that the delta2,3-2-acetamido-2-deoxy-D-glucoseen at the reducing end of the unsaturated tetrasaccharide has a half-chair conformation. This conformation is similar to the one proposed for the sugar at subsite D in the lysozyme-substrate complex in the transition state. Addition of the unsaturated tetrasaccharide to a solution of hen egg white lysozyme quenched the fluorescence of the enzyme and shifted the fluorescence maximum to the blue, similar to the effect produced by the parent compound. The association constant of the unsaturated tetrasaccharide and lysozyme was measured at pH 6.0 and 24 degrees by spectrofluorimetry and microcalorimetry and found to be 1.45 X 10(5) M-1 and 2.5 X 10(5) M-1, respectively. The average value is 100 times higher than that found for the binding of unmodified tetrasaccharide to the enzyme under the same conditions. The unsaturated tetrasaccharide proved to be a better inhibitor of the lysis of Micrococcus luteus cells than the parent compound by a factor of 35. These results support the hypothesis that the active site of the enzyme is constructed so as to bind the transition state for the reaction it catalyzes more firmly than the substrate itself.

Animals↗
PubMed1976

Saccharides on teratocarcinoma cell plasma membranes. Their investigation with radioactively labelled lectins.

We have studied the interaction of five lectins differing in their sugar specificity, with the surface of clonal cell lines derived from transplantable murine teratocarcinoma. The results show that the differentiation from primitive embryonal carcinoma cells into parietal yolk sac cells is accompanied by changes in cell surface saccharides. These changes consist of a marked decrease in the total number of binding sites for the L-fucose-specific lectin of Lotus tetragonolobus and a large increase in the total number of binding sites for wax bean agglutinin. It is suggested that these differences can be used as markers in the study of this early embryonic differentiation. No agglutination of primitive embryonal carcinoma cells or of parietal yolk sac cells by low concentrations (10mug/ml) of concanavalin A, soybean agglutinin or the fucose binding proteins was observed.

Binding Sites↗
PubMed1976

The binding of glycoconjugates to human-milk D-galactosyltransferase.

Through the use of affinity chromatography, a homogeneous preparation of human beta(1 leads to 4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human alpha-lactalbumin. A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and the beta(1 leads to 4) linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor moleucles. Furthermore, experiments with Na-acetylglucosamine-beta(1 leads to4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent Km values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, alpha-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides. Although alpha-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-glucose resulting in the production of lactose, no transfer of galactose was observed to beta(1 leads to 4)-linked glycose oligomers or to a collagen glycopeptide, D-glycopyranosyl-alpha(1 leads to 2)-D-galactopyranosyloxy-beta(1 leads to 5)-lysine. IT therefore appears that alpha-lactalbumin can only modify human A protein for monosaccharide acceptors.

Acetylglucosamine↗
PubMed1976

The requirement for tetravalency of soybean agglutinin for induction of mitogenic stimulation of lymphocytes.

The mitogenic activity of soybean agglutinin was found to depend on the presence of lectin aggregates formed in lectin preparations stored in the lyophilized state. Such soybean agglutinin preparations gave maximal stimulation of untreated pig lymph node cells and neuraminidase-treated mouse spleen cells at relatively high concentrations, ranging from 100 to 2000 mug/ml. After separation into unaggregated (divalent) and polymeric (tetra-and multivalent) fractions, it was found that the unaggregated lectin did not stimulate the cells, while the tetravalent and multivalent fractions were active and gave maximal stimulation at a concentration of 10 mug/ml. These results suggest that soybean agglutinin must have at least four sugar binding sites in order to be able to stimulate lymphocytes.

Acetylgalactosamine↗