Membrane-associated carbohydrate-binding molecules appear to be involved in a variety of physiologically significant interactions. Galaptins, a class of endogenous well-conserved, beta- galactoside-binding lectins, appear to play a role in growth control, cell adhesion and metastatic phenomena and immune modulation. Galaptin appears to be ubiquitous in human tissues but is differentially expressed in human leucocytes. It is the purpose of this project to carry out the physico- and immunochemical characterization of galaptin isolated from human buffy coat cells. Galaptin will be isolated from normal human peripheral leucocytes by asialofetuin-Sepharose affinity chromatography. The purity, molecular weight and subunit composition will be analyzed by single and 2-D electrophoretic procedures and by Western blotting followed by immunodetection. Native galaptin will be analyzed by FPLC gel filtration. Physico- chemical characterization of galaptin will include amino acid composition and amino acid sequence analyses. Compositional studies will be carried out on a Waters Pico-Tag system. Sequence studies will be carried out on galaptin degradation fragments with an Applied Biosystems 470A sequenator. The specificity of anti-galaptin rabbit sera in hand will be determined. Galaptin carbohydrate-binding specificity will be characterize utilizing ELISA and affinity bead methodologies. Binding-site probes will include well-characterize glycoconjugates and their degradation products as well as a large battery of completely characterized synthetic carbohydrate compounds. The distribution of galaptin among leucocyte subtypes will be determined by immunohistochemical procedures utilizing our anti- galaptin sera. The research described in this application will lead to the physico-chemical characterization of a carbohydrate-biding protein present in human peripheral leucocytes and identification of cells of origin. These studies will lay the necessary foundation to pursue the elucidation of carbohydrate-binding protein function and how pertubation of function affects cell behavior.
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