Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid

Dev, Shemul (2014) Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid. Masters thesis, Memorial University of Newfoundland.

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Abstract

The sea urchin major yolk protein (MYP) is localized in nutritive phagocytes in the testis and ovary, as well as the egg and coelomic fluid of the adult sea urchin. We and others have shown that the egg MYP can drive calcium-dependent, membrane-membrane interactions. However, much less is known about the coelomic fluid-localized MYP. Therefore, we have begun a comparative biochemical analysis of the egg and coelomic fluid MYPs. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg and coelomic fluid. Under reducing conditions, there were two species of MYP both in the egg and the coelomic fluid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the egg and coelomic fluid MYPs under reducing and non-reducing conditions revealed that under reducing conditions, there are two species in each fraction, 170- nad 240 kDa in the egg and 180- and 250 kDa in the coelomic fluid. However, under non-reducing conditions only one species is present in each fraction, 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 protease peptide mapping showed that all four polypeptide species have very similar primary structures. Further analysis with circular dichroism (CD) revealed that the purified 170- and 180 kDa species possess different secondary structural features. Endogenous tryptophan fluorescence measurements in the presence of different concentrations of calcium showed a notable difference in apparent dissociation constants (calcium); 245- and 475 μM for the egg and coelomic fluid MYPs, respectively. Interestingly, there was no notable difference in the apparent dissociation constants for zinc. Additional assays revealed that both the 170- and 180 kDa species have different calcium requirements for binding to liposomal membranes, with an apparent dissociation constants (calcium) of 10- and 290 μM for the 170- and 180 kDa species, respectively. In addition, both the 170- and 180 kDa species have different calcium requirement to induce vesicular aggregation, which is correlated with the calcium induced tertiary structural change in both polypeptides. Collectively, these results identify structural differences between the egg and coelomic fluid MYPs which may reflect different functional capabilities between these species.

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