Binding of divalent metal ions to hepatic soluble /]-galactoside binding lectin was studied using electron spin resonance (ESR) spectroscopy. The Mn 2+ bound to hepatic lectin could be displaced by Mg 2+, Cu 2+, Ni 2+ and Ca 2+ but not by Sr 2+. As the ionic radii of Mg 2+ (0.65 A), Cu 2+ (0~73 ~.) and Ni 2+ (0.72 A) are appreciably smaller than Ca 2+ (0.99 A), it appears that the Mn 2+ binding site is more accessible to Mg 2+, Cu 2+, and Ni 2+ as compared to Ca 2+, the ionic radius of Mn 2+ being 0.80 .~,. Sr 2+ with an ionic radius of 1.13 is thus unable to displace bound Mn 2+. Surprisingly, the presence of specific sugars like a-lactose, or a-o-galactose facilitated the displacement of bound Mn 2+ by metal ions whereas non-specific sugars, i.e. a-D-glucose, j~-ofructose and a-D-ribose had no effect. It appears that minor perturbations in the saccharide binding site significantly affect the ability of the metal binding site to ligate bivalent metals.
SiaIic acid is now known to serve as ligand for lymphocyte lectins known as selectins. Although its role as a ligand for adhesion molecules has been studied extensively, no studies have been performed to determine the physiological changes in lymphocytes after binding of sialic acid to lymphocyte lectin. We report for the first time that interaction of lymphocytes with siahc acid severely restricts the rotational mobility of the cell surface proteins as well as membrane lipids, as studied by EPR spectroscopy using spin probes. Binding of mucin totally immobilizes the lymphocyte membrane. Surprisingly the binding of sialic acid or mucin also immobilized the aqueous probe TEMPO, indicating an appreciable increase in cytoplasmic viscosity.
This paper reports for the first time, that binding of various mono-, di-, and trisaccharides to membrane lectins reduces the rotational motion of membrane proteins and lipids indicating a decrease in membrane fluidity as studied by EPR spectroscopy using spin probes. Interaction of polysaccharides with lymphocyte resulted in an extensive decrease in membrane fluidity making the membrane almost rigid. The decrease in fluidity was dose-dependent, dependent on the multivalency of the iigand used, and was sensitive to presence of EDTA and sodium azide. Binding of two different carbohydrate ligands on their respective surface iectins has a synergistic effect on the decrease in membrane fluidity.used as probes for the hydrocarbon portion of the membrane bilayer. TEMPO was used as a probe for the aqueous compartments while MECP was used to probe the surface portion with reactive NH2 group. EPR spectra obtained were analysed by computing the rotational correlation time (T~). Tc is the measure of degree of immobilization of the spin label, hence a measure of the local viscocity. Our hypothesis is that the specific binding of carbonhydrates to membrane surface lectins (selectins) induces an energy-dependent stiffening of the lipid membrane.
Materials and methods
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