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Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

At least 181 records · Page 10Linked to original sources
PubMed1978

Separation and biological properties of Phaseolus vulgaris isolectins.

Two isolectins (L4E0-PHA and L0E4-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L4E0-PHA isolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L0E4-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a higo lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L0E4-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer - specific oligosaccharide, are not involved in the process inducing mitosis.

Agglutination↗
PubMed1978

Ligands containing heavy atoms: perturbation of phosphorescence of a tryptophan residue in the binding site of wheat germ agglutinin.

Information on the structure of binding sites of wheat germ agglutinin was obtained on the basis of fluorescence and phosphorescence changes of tryptophan residues induced by the binding of several thiomercuribenzoate derivatives of glycosides. The thiomercuribenzoate derivatives bind selectively to wheat germ agglutinin in the same way as the corresponding sugars. Using the thiomercuribenzoate of di-N-acetyl-beta-chitobiose, it was found that: (i) the fluorescence of tryptophan residues was drastically quenched at both 298 and 77 K; (ii) the phosphorescence intensity was strongly enhanced at 77 K; (iii) the phosphorescence lifetime was markedly decreased. A similar effect was observed with the thiomercuribenzoate of N-acetyl-beta-D-glucosamine. These changes were completely reversed upon addition of 1-O-methyl-di-N-acetyl-beta-chitobioside. The thiomercuribenzoate of beta-D-glucose had no effect at all, and the thiomercuribenzoate of tri-N-acetyl-beta-chitotriose had a limited effect. These results are interpreted as a specific heavy atom effect due to a close contact between one tryptophan residue of the protein and the heavy atom of the bound ligand. They are consistent with the view that: (i) binding sites of wheat germ agglutinin may be divided in three subsites, A, B, and C; (ii) a tryptophan residue is in the binding site at subsite C; and (iii) this residue and the ligand are in close contact. This new method, using the enhancement of spin-orbit coupling due to the selective perturbation induced in a tryptophan residue by a ligand containing a heavy atom, has proved to be suitable for locating the tryptophan residue in the binding site of wheat germ agglutinin and can probably be extended to other sugar-binding proteins.

Binding Sites↗
PubMed1977

Limulin (Limulus polyphemus lectin): mitogenic effect on human peripheral lymphocytes.

The lectin from hemolymph of Limulus polyphemus was purified by affinity chromatography on insolubilized bovine submaxillary mucin. The purity of the protein was checked by crossed immunoelectrophoresis. Agglutination of human red blood cells was completely abolished after neuraminidase treatment, while other cells were still agglutinable after the same treatment but required a higher concentration of lectin. Limulin was able to simulate about 50% of human peripheral lymphocytes. This mitogenic effect could be inhibited by bovine submaxillary mucin but not by the disialylated mucin. Related to the known oligosaccharide-binding specificity of limulin and of the other nonspecific activators of lymphocytes, the authors suggest that lymphocyte stimulation is triggered by binding to a glyco-conjugate bearing the following carbohydrate chains: NANA leads to GalNac leads to or NANA leads to Gal leads to GlcNAc leads to Man.

Agglutinins↗
PubMed1977

Ultrastructural visualization of cellular carbohydrate components by means of lectins on ultrathin glycol methacrylate sections.

A method for the visualization of cellular carbohydrate components by both light and electron microscopy using lectins on glycol methacrylate sections is proposed. This method, which is an application of the lectin-peroxidase affinity technique, solves the problem of limited penetration when it is attempted to demonstrated lectins receptors within the tissue block. Following partial dissolution of glycol methacrylate from thin sections using alcohol, they are incubated successively with lectin (Concanavalin A or wheat germ agglutinin), horseradish peroxidase (Sigma, type II), 3-3' diaminobenzidine and H2O2 and then with OsO4-Different kinds of tissues and cells have been used to test the method: mouse myocardium, rat epididymis, a protozoon Gregarina blaberae and the bacterium Escherichia coli. The localization of carbohydrate residues deomonstrated by this method within the different tissues and cells is consistent with the findings from other published studies. Controls have been performed (i.e., omission of the lectin, lectin and its inhibitor) and these demonstrate the specificity of the method.

Animals↗
PubMed1976

[Demonstration of an endo-beta-N-acetyl-glucosaminidase in rabbit serum].

An endo-beta-N-acetyl glucosaminidase of rabbit serum has been characterized after purification by affinity chromatography. The specificity was determined by fluorimetric monitoring of the hydrolysis of 4-methylumbelliferyl-beta-glycosides and by thin layer chromatography identification of the hydrolysis products of fluoresceinyl-beta-glycosides. This enzyme acts on the following oligosaccharides GlcNac-beta-1-4 leads to GlcNAc-beta-O-R giving free di-N-acetyl chitobiose and R-OH as hydrolytic products.

Acetylglucosaminidase↗
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

Nanosecond-pulse fluorimetry of wheat-germ agglutinin (lectin).

Nanosecond-pulse fluorimetry of wheat germ agglutinin is analyzed as a function of both excitation and emission wavelengths. When excited at 280 nm, wheat germ agglutinin fluorescence exhibited three lifetimes: one corresponding to the tyrosine residues as a whole and two others corresponding to the tryptophyl emission. The tyrosine contribution to the emission spectrum deduced from this method was in good agreement with that reported previously in steady-state fluorescence experiments [Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555-567]. The fluorescence decay of each tryptophan residue was not a single exponential function when wheat germ agglutinin was excited at 295 nm. This could be related to the microenvironment of the indole chromophores in the protein. The comparison of the quantum yield and of average lifetime showed that some tryptophan residues were completely quenched. Energy transfer from tyrosines to tryptophan residues previously detected in steady-state fluorescence was also revealed by fluorescence decay measurements. Comparison of both methods showed that an important part of transfers occurred with a very fast rate equal to or greater than 10(10) s-1. Both lifetimes and the ratio of the short and the long-lived component were found dependent on tri-N-acetylchitotriose binding.

Binding Sites↗
PubMed1976

An improved method for purification of wheat germ agglutinin (lectin) by affinity chromatography.

A simple purification of wheat germ agglutinin from commercial wheat germ is described. From defatted ground wheat germ, the lectin was extracted and then purified in a single step by filtration on an ion exchange chromatography column and adsorption on an insolubilized N-acetyl glucosamine derivative. The amount of lectin obtained from 1,000 g of wheat germ was larger than 500 mg. Although the yield was at least twice higher than that obtained with other methods, no impurities could be detected, and molecular characteristics are in good agreement with the protein purified by more sophisticated procedures.

Amino Acids↗
PubMed1975

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.

Binding Sites↗