RESUMO
The major yolk protein (MYP) is localized to the egg and coelomic fluid of the adult sea urchin. While the egg-localized MYP has been extensively studied, much less is known about the coelomic fluid-localized protein. Therefore, we have conducted a comparative biochemical analysis of these proteins. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg, 170- and 240 kDa, and the coelomic fluid, 180- and 250 kDa. Fractionation in polyacrylamide gels revealed that under reducing conditions both species were present in each location. However, in the absence of reducing agent only one species was present in each fraction: 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 peptide mapping indicated that all four species have very similar primary structures. Circular dichroic spectral analysis and endogenous tryptophan measurements of the purified 170- and 180 kDa species revealed distinctive secondary and tertiary structural features with notable differences in their responses to calcium: apparent Kds of 245- and 475 µmol/L were measured for the 170- and 180 kDa species, respectively. Further analysis revealed that both species have differing calcium requirements for binding to membranes as well as protein-dependent, membrane-membrane interaction. We discuss the functional implications arising from the structural differences which exist between the egg and coelomic fluid resident MYPs.
