Parvalbumin characterization from the euryhaline stingray Dasyatis sabina

Abstract

The Atlantic stingray, Dasyatis sabina found along the Gulf of Mexico and southeastern Atlantic coasts, is a euryhaline species of elasmobranch. This species is able to osmotically compensate for changing environmental salinity by altering plasma and intracellular solutes, including urea and counteracting methylamines (betaine and TMAO). Parvalbumin (PV) is an intracellular protein that facilitates muscle relaxation by sequestering calcium. Determining the effects that in situ concentrations of urea (146 mM), betaine (62 mM), and TMAO (11 mM) have on PV function in marine and freshwater adapted populations of D. sabina could provide insight into intracellular correlates of euryhaline tolerance for this species. PV from marine and freshwater populations of D. sabina was identified and purified by SDS-PAGE, western blot analysis, and full amino acid sequence analysis. Both populations exhibited two PV isoforms, PV I (approximately 12.18 kDa mw) and PV II (11.96 kDa mw). PV dissociation constants (K(D)) were determined in the presence and absence of physiological concentrations of urea, betaine, and TMAO by fluorescence spectroscopy using the fluorescent Ca(2+) indicator fluo-3 which competes with PV for Ca(2+). Functional studies revealed PV I showed no significant changes in calcium binding from in situ muscle conditions, except in the presence of betaine. In contrast, PV II's ability to bind calcium was increased relative to physiological conditions in the presence of each osmolyte independently. Thus, it appears that organic osmolytes have isoform specific effects on PV function.