This laboratory has been using a derivatized bead assay to study cell surface properties in many experimental systems. Here we test its efficacy in the yeast/concanavalin A (Con A) binding model to determine if immobilized Con A is inhibited by the same saccharides that inhibit free Con A binding. This system offers an excellent test case for the efficacy and validity of the derivatized bead assay. Con A binds to yeast because yeast are rich in cell surface mannose residues, a Con A preferential binding sugar. Here we examine the effects of 30 different sugars (0.05 M) on the binding of yeast to agarose beads derivatized with Con A, in a total of 3998 trials, with an average of over 133 replicates for each sugar. The most inhibitory sugars included D(+) melezitose, D(+) trehalose and maltotriose, the same sugars that effectively inhibit binding of free Con A. The results suggest that the bead assay is an effective approach to study the binding properties of cells, and most important, it is so rapid that hundreds of trials can be done in the time it would take to do one trial using conventional assays (supported by NIH NIGMS SCORE, RISE, MARC, the ITQ program, and the Joseph Drown Foundation).
We developed a novel method to probe mechanisms of cellular interactions by microdissecting the archenteron and blastocoel roof out of 48–54 hr Lytechinus pictus sea urchin ermbryos. These are the two components of a classic cellular interaction in the NIH designated model sea urchin embryo. Here we test the ability of isolated fixed archenterons to bind to agarose beads derivatized with concanavalin A (Con A) in artificial sea water (ASW) at pH 4,6,8 (control), 9 or 10 or in the presence of 4M NaCl or 2M or 4M alpha methyl glucose. The binding of a total of 204 isolated archenterons was only inhibited in 4M alpha methyl glucose (p less than 0.05 compared with controls). All other conditions gave p values of greater than 0.05. As Con A beads bound to the isolated archenterons and alpha methyl glucose inhibited the interaction, the results suggest that Con A binding ligands are present on the isolated archenterons. While 4M sugar is high, the archenterons were fixed, reducing osmolarity concerns. High monosaccharide concentrations are often required to inhibit lectin‐involved binding interactions. Part I of this series provides evidence for glycan involvement in the actual cellular interaction. This microdissection method allows probing of isolated components of cellular interactions outside of the embryo proper, to avoid complex problems that may occur in whole embryos (supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).
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