This research proposal is concerned with studies on carbohydrate-protein interaction. Primary emphasis will be on carbohydrate-lectin interaction. A decapeptide containing a cysteine residue involved both in carbohydrate and metal binding, and homologous to a metal binding region in concanavalin A, has been isolated from the lima bean lectin. It has been synthesized in amounts sufficient to study its metal binding properties (by CD spectroscopy and metal chelate affinity chromatography) and to prepare antibodies to assay for homologous regions in other legume lectins. A series of carbohydrates (chitobiose, lactosamine, N-acetyl-D-galactosamine) and hydrophobic probes, containing spin labels in strategic positions, are being synthesized and will be studied by electron spin resonance spectroscopy for their capacity to bind to, and elucidate the environment of, the carbohydrate and hydrophobic binding sites of lectins. Lectin-carbohydrate complexation will also be studied by NMR spectroscopy. Initially, the interaction between N,N'-diacetylchitobiose and N-acetyllactosamine with the N-acetyl-D-glucosamine binding lectin from Datura stramonium seeds will be studied. The sugar binding specificity and biological properties of a number of new lectins will be investigated in order to discover new sugar binding specificities which should prove useful for structural studies on glycoproteins and glycolipids. A new approach to labeling the sugar binding sites of lectins involves affinity labeling in the bound state. Termed solid phase affinity labeling, it should allow covalent labeling of lectin active sites in good yield. A new approach for using fluorescein-labeled lectins in flow cytometry involves use of a functionally monovalent lectin; the 3 A subunit sites of the Griffonia simplicifolia A3B isolectin will be saturated with the haptenic sugar N-acetyl-D-galactosamine allowing inter- reaction of the remaining Alpha-D-galactosyl binding B subunit with reactive cells, e.g. Ehrlich ascites tumor cells. Antibodies to 2-0-Alpha-D-glucopyranosyl-D-galactose, the disaccharide unit of collagen will be raised in rabbits and used to label type IV membrane collagen. The lima bean lectin gene will be cloned and translated; site-specific mutagenesis should allow identification of amino acid residues involved in carbohydrate binding.
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