Biochemical analysis of a Ca2+-dependent membrane-membrane interaction mediated by the sea urchin yolk granule protein, toposome.
Toposome, a high molecular mass protein, is an abundant component of the yolk granule in the sea urchin egg and embryo. Toposome is composed of a 160 kDa polypeptide that is proteolytically processed into smaller species of 120 and 90 kDa during embryonic development. The exact biological function of toposome during early development is unknown. In this study we have examined calcium binding to toposome and the effect of this binding on the secondary and tertiary structural characteristics of the purified protein. Initially, we used equilibrium dialysis to quantify calcium binding to toposome. Monophasic binding of up to 600 M of calcium per mole of protein was detected with an intrinsic dissociation constant (calcium) of 240 microm. Increasing concentrations of calcium resulted in an increase in alpha helical content from 3.0 to 22.0%, which occurred with an apparent dissociation constant (calcium) of 25 microm. In parallel experiments, toposome binding to liposomes required similar concentrations of calcium; an apparent dissociation constant (calcium) of 25 microm was recorded. Endogenous tryptophan fluorescence measurements, both in the presence and absence of liposomes, demonstrated that the tertiary structure is sensitive to increasing concentrations of calcium with an apparent dissociation constant (calcium) of 240 microm. Toposome-driven, liposome aggregation assays revealed a similar calcium requirement. Collectively, these results define a two-step model for calcium modulation of toposome structure and function.
Hayley
Michael
M
Department of Biochemistry, Memorial University of Newfoundland, St John's, Canada.
Perera
Aruni
A
Robinson
John J
JJ
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004527:Egg Proteins / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008566:Membranes / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D017433:Protein Structure, Secondary / Q000187:drug effects; D012617:Sea Urchins / Q000737:chemistry*
D004527:Egg Proteins; D006023:Glycoproteins; D008081:Liposomes; C118907:toposome glycoprotein complex; D002118:Calcium