Thesis

Quantitative evaluation of characterized alpha and beta glycosidase effects on sea urchin embryo cellular interactions

Using the NIH designated sea urchin embryo model, we are studying the molecular basis of a set of cellular interactions that have interested investigators for over a century, archenteron organization/elongation/attachment to the blastocoel roof. Here we evaluate the effects of four commercially available glycosidases: alpha and beta amylase and alpha and beta glucosidase, that we independently characterized, on embryo development. In quantitative dose-response experiments we show an I-50 of 10-30 units/ml, in artificial sea water or low calcium artificial sea water, of beta amylase and alpha glucosidase in inhibiting archenteron organization/elongation/attachment to the blastocoel roof in living 48 hr Lytechinus pictus gastrula embryos using a microplate assay that allows quantitative assessment of embryo morphological characteristics. All concentrations tested (up to 5000 units/ml) of alpha amylase and beta glucosidase had no significant effects on archenteron morphology. All experiments involved assessing 3600-5600 embryos in at least 144 replicate wells for each enzyme. The differences in archenteron XVll morphology compared with controls were significant with p values of less than 0.05, while any differences observed with the ineffective enzymes had p values of greater than 0.05, using two-tailed t-tests. All enzymes used were tested for their activity on known substrates, and found to be active, and specific unit activities were established. Product inhibition studies suggested that the effective enzymes acted by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectible protease activity in the enzyme samples. The results suggest that glycans are involved in these cellular interactions and we discuss possible conformations of these glycans.

Le relazioni

Elementi