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Shemul Dev - Feb 1

Comparative Biochemical Analysis of the Major Yolk Protein in the Sea Urchin Egg and Coelomic Fluid

The sea urchin major yolk protein (MYP) has been previously considered to have a vitellogenin (Vg) like role to supply nutrients to the developing embryos and larvae. However, neither MYP has any sequence homology with Vg proteins nor it is female specific (present in both male and female) in sea urchin. Major yolk protein is localized in the testes, ovary and also present in coelomic fluid of the adult sea urchin. We and others have shown that egg MYP can drive Ca2+- dependent, membrane-membrane interactions. But much less is known about the coelomic fluid localized MYP (CF MYP). Therefore, we have begun a comparative biochemical analysis of the egg and CF MYPs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of egg and CF MYP enriched fractions under reducing and non-reducing conditions revealed that, under reducing condition, there are two species in each fraction (i.e; for egg 240 kDa and 170 kDa; for CF 250 kDa and 180 kDa) but under non-reducing conditions only one species is present in each fraction (i.e; for egg 240 kDa; and for CF 250 kDa). In addition, V8 protease peptide mapping showed that all four polypeptide species have very similar primary structures. Furthermore, sucrose density gradient ultracentrifugation (15-30 %) revealed unique elution profiles for each of the four species. This result suggested that each of the four polypeptides is unique in their size and/ or shape. Further analysis with CD confirmed that the 170 kDa (egg) and 180 kDa (CF) species are different in their secondary structure. Endogenous tryptophane fluorescence measurement in the presence of different concentration of Ca2+ shows a notable difference in apparent dissociation constant of 225 µM and 500 µM for Egg MYP (170 KDa) and CF MYP (180 KDa), respectively. Collectively, these results identify structural differences between the egg and CF MYPs hence may result in different functional capabilities between these species.

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