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A Protein in Normal Nurse Shark Serum Which Reacts Specifically with Fructosans

I. Purification and Immunochemical Characterization^1,2,

From the Departments of Microbiology, Neurology, and Human Genetics and Development, College of Physicians and Surgeons, Columbia University, and the Neurological Institute, Presbyterian Hospital, New York 10032 and the Department of Microbiology, University of Miami, Florida 33155

Abstract

A protein which specifically precipitates with the fructosans, levans and inulin, was found in most normal nurse shark sera. It could be specifically purified on insoluble immunoadsorbent columns by using Sephadex G-75 or levan gel. From quantitative precipitin and inhibition studies, the combining site was found to react specifically with the -D-fructofuranosyl (2 1) -D-fructofuranosyl and -D-fructofuranosyl (2 6) -D-glucopyranosyl residues. The best inhibitor was a tetrasaccharide, -D-fructofuranosyl (2 1) -D-fructofuranosyl (2 1) -D-fructofuranosyl (2 6) D-glucose (3F1G). The protein was unrelated to shark IgM antigenically and appears not to be an immunoglobulin of the usual type. Binding of the protein with its specific ligands caused specific fluorescence quenching and the association constants for various mono- and oligosaccharides were measured. The calculated relative association constants from precipitin inhibition assays and from fluorescence quenching for various mono- and oligosaccharides agreed closely and the homogeneity of the combining site was indicated by both methods. The association constant (K₀) for 3F1G was 3.7 x 10⁴ 1/M. Two other carbohydrate-reacting proteins were also found in normal nurse shark sera. One precipitated with perennial rye grass levan which contained 2 6 linked fructofuranosyl residues. The other was specific for dextran B1355-S-4, which contained a high percentage of (1 3) linked glucopyranosyl residues.

Footnotes

¹ Aided by grants from the National Science Foundation (GB-8341 and GB-25686) to Dr. E. A. Kabat and a General Research Support grant of the United States Public Health Service to Columbia University; and the United States Public Health Service Research Grant (AI-05758) from the National Institute of Allergy and Infectious Disease to the University of Miami (Dr. M. M. Sigel).

² From Part I of a dissertation submitted by V. Harisdangkul in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University, New York.

³ Rockefeller Foundation Fellow, 1967 to 1971.