Of concanavalin

Figure 26.6 (a) The /3-pleated sheet secondary structure of proteins is stabilized by hydrogen bonds between parallel or antiparallel chains, (b) The structure of concanavalin A, a protein with extensive regions of antiparallel / sheets, shown as flat ribbons. 

S. Fortier, M. Touaibia, S. Lord-Dufour, J. Galipeau, R. Roy, and B. Annabi, Tetra- and hexavalent mannosides inhibit the pro-apoptotic, antiproliferative and cell surface clustering effects of concanavalin A Impact on MT1-MMP functions in marrow-derived mesenchymal stromal cells, Glycobiology, 18 (2008) 195-204. 

M. Sleiman, A. Varrot, J.-M. Raimundo, M. Gingras, and P. G. Goekjian, Glycosylated asterisks are among the most potent low valency inducers of Concanavalin A aggregation, Chem. Commun. (2008) 6507-6509. 

Multiple-ion monitoring is, however, of considerable value in structural studies, but only if model compounds of known structure are available for comparison. Such an approach has been used in the study of the carbohydrate structures of glycoproteins from different tissues.50 Separation of glycopeptides obtained from various tissues was performed on columns of concanavalin A-Sepharose. Structural analysis by multiple-ion monitoring of partially methylated, alditol acetates derived from the various fractions indicated that the glycopeptides were separated according to the linkage pattern of mannose (see Fig. 1). 

K. R. Srinivasan, S. Mansouri, and J. S. Schultz, Coupling of concanavalin A to cellulose hollow fibers for use in glucose affinity sensor, Biotechnol. Bioeng. 23, 233 (1986). 

I. J. Goldstein and R. N. Iyer, Interaction of concanavalin A, a phytohemagglutinin, with model substrates, Biochim. Biophys. Acta, 121 (1966) 197-200. 

K Shimura, K Kasai. Determination of the affinity constants of concanavalin A for monosaccharides by fluorescence affinity probe capillary electrophoresis. Anal Biochem 227 186-194, 1995. 

Lin, S.-L., Stern, E. A., Kalb (Gilboa), A. J., and Zhang, Y. (1990). Evidence from X-ray absorption fine structure spectroscopy for significant differences in the structure of concanavalin A in solution and in the crystal. Biochemistry, 29, 3599-3603. 

Derewenda Z, Yariv J, Helliwell JR, Kalb AJ, Dodson EJ, Papiz MZ, Wan T, Campbell J. The structure of the saccharide-binding site of concanavalin A. EMBO J 1989 8 2189-2193. 

For identification the lectins must be labeled. The best labels are biotin (streptavidin-enzyme conjugate) or digoxigenin (anti-digoxigenin antibody-enzyme conjugate), but in the case of Concanavalin A, direct labeling with HRP is possible (see below). 

Figure 27. Comparison of the L-Clu-induced integrated channel currents between two different preparations of GluR-incorporated BLMs upon injecting the identical concentration of l-GIu (0.10 nM). Applied potential +50 mV. Conditions 9.8 mM HEPES-NaOH (pH 7.6) containing 0.52 M NaCI, 0.19 mM CaCl2, 4.8 mM glycine, 24 pg mL of concanavalin A, 8.1 mM sucrose and 0.40 M formamide in both cis and trans side solutions. Proteoliposomes were injected only to the cis side and agonist solution was added to the trans side. Applied potential +50 mV. The l-GIu solution was injected to the trans side. ...

Figure 28. Changes in the integrated channel currents for multichannel BLM upon addition of L-Clu, MK-801, and DNQX in series. Solution condition (a) the same as in Figure 27, (b) 20 mM citric acid, 42 mM Tris, 0.15 M NaCI, 5.0 mM glycine, 0.20 mM CaCb, and 25 mg ml" of concanavalin A, pH 7.6, and (c) the same as in Figure 27. Applied potential (a) +30 mV, (b) -30 mV, and (c) +30 mV. The L-Clu solution was injected to the trans side. The antagonist was injected to both sides.

The structure of the (l- 2)-a-D-mannopyranan has been confirmed by methylation analysis, and its interaction with concanavalin A has been tested. Concanavalin A forms a weak complex with this oligosaccharide, and this is precipitated. Two molecules of concanavalin A must, therefore, be able to link to a sequence of two, or three, (1— 2)-a-D-mannopyranosyl residues. The polymer thus gives some insight into the steric requirements, and the size of the active site, of this lectin.120... 

Biochemical studies have also suggested an asymmetric orientation of constituents in lipid globule membrane. By comparison of specific activities of enzymes in washed lipid globules and released membrane, Patton and Trams (1971) suggested that the active site of Mg2+-adeno-sine triphosphatase was accessible to substrates on both faces of the membrane and that of 5 -nucleotidase on the outer membrane face. Recent evidence from studies of Concanavalin A inhibition of globule membrane and plasma membrane 5 -nucleotidase support an outer surface localization for the active site of this enzyme (Carraway and Carra-way 1976 Snow et al. 1980). Kobylka and Carraway (1973) observed that exposure of lipid globules to proteolytic enzymes resulted in cleavage of all major membrane-associated proteins. They concluded that... 

Patton, S. and Hubert, J. 1983. Binding of Concanavalin A to milk fat globules and release of the lectin-membrane complex by Triton X-100. J. Dairy Sci. 66, 2312-2319. 

Figure 23-3 Infrared absorbance spectra of the amide regions of proteins. (A) Spectra of insulin fibrils illustrating dichroism. Solid line, electric vector parallel to fibril axis broken line, electric vector perpendicular to fibril axis. From Burke and Rougvie.24 Courtesy of Malcolm Rougvie. See also Box 29-E. (B) Fourier transform infrared (FTIR) spectra of two soluble proteins in aqueous solution obtained after subtraction of the background H20 absorption. The spectrum of myoglobin, a predominantly a-helical protein, is shown as a continuous line. That of concanavalin A, a predominantly (3-sheet containing protein, is shown as a broken line. From Haris and Chapman.14 Courtesy of Dennis Chapman.

Depending on the fit, the number and strength of contacts, and other factors, a monomer-oligomer equilibrium will exist. For example, in the case of Concanavalin A the tetramer is in equilibrium with dimer and monomer under normal conditions. In the case of insulin the monomer, dimer, and hexamer are all in equilibrium. In hemoglobin the dimer and tetramer are in equilibrium. In the case of the adsorption of such proteins, one must not only know what is the aggregation state of the protein in solution, but must be able to deduce the adsorbed state. Do the molecules adsorb as dimers 21) Do they adsorb as dimers and then associate as tetramers or hexamers on the surface, etc. ... 

Four different amounts of the four gangliosides were added to microtiter wells containing human PBMC s and 18 micrograms of concanavalin-A. All four gangliosides markedly inhibited lymphoblastic transformation when 12.6 nanomoles was added to the cultures with a total volume of 0.2 ml (Table II). As little as 1.6-nanomoles of GDlb and GTlb resulted in substantial inhibition, while much less inhibition occurred with equivalent amounts of GM1 and GDla. 

Nishihata, T., and T. Higuchi. 1984. Promoting effect of concanavalin A on transport of sodium cefoxitin and phenol red from rat rectal compartment. Life Sci 34 419. 

Goldstein L, Hollerman C, Smith EE. Protein-carbohydrate interaction. II. Inhibition studies on interaction of concanavalin A with polysaccharides. Biochemistry 1965, 4, 876-883. 

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