P1.70 Jan. 4 Parvalbumin identification and functionality in the freshwater and marine Atlantic stingray, Dasyatis sabina HEFFRON, Jennifer K.*; MOERLAND, Timothy S.; Florida State University; Florida State University firstname.lastname@example.org
The Atlantic stingray, Dasyatis sabina , found along the Gulf of Mexico and southeastern Atlantic coasts, is an unusual elasmobranch in its ability to handle variations in environmental salinity. When faced with an increase in salinity this species is able to osmotically compensate by increasing plasma and intracellular solutes, including urea and the counteracting methylamines (betaine and TMAO). Parvalbumin (PV) is an intracellular protein that facilitates muscle relaxation by sequestering calcium. Determining the effects that urea, betaine and TMAO concentrations have on PV in both the marine and freshwater species of D. sabina could provide insight into these species unique adaptations to their environment. D. sabina acclimatized to fresh water was collected by hook and line from Lake Monroe, located in the St. Johns River System in Florida. This represents the only permanent freshwater population of elasmobranchs in North America. Representatives from the marine environment were collected by gillnet in the Gulf of Mexico. PV from both marine and freshwater D. sabina were purified by gel permeation and DEAE chromatography. Identity and purity were confirmed by SDS-PAGE, western blot analysis, and N-terminal sequence analysis. Both exhibited two PV isoforms, a major form (13.37 kDa mw) and a minor form (12.35 kDa mw). Preliminary parvalbumin dissociation constants (KD) were determined at physiological concentrations of urea, betaine, and TMAO by fluorescence spectroscopy using the fluorescent Ca2+ indicator fluo-3, which competes with PV for Ca2+. KD was determined to be approximately 10.12 nM for the major PV isoform and 12.70 nM for the minor PV isoform.