The physiological properties of muscles, such as contraction and relaxation, are highly dependent on isoforms of various muscle proteins.  Different muscle fibre types display different physiological properties due to variations in the isoform(s) of myosin, troponin and other myofibrillar proteins.  Parvalbumin, a myoplasmic protein with two isoforms, is a low molecular weight protein (9-11 kD) that appears to aid in relaxation.  It does so by binding to free Ca2+, which reduces the intracellular concentration of the ion. Since calcium plays a necessary role in contraction by binding to the regulatory protein troponin, a decrease in its intracellular concentration will result in relaxation of the muscle fiber.  For parvalbumin to bind Ca2+, Mg2+ must not be bound. Furthermore, the dissociation rate for Mg2+ is believed to determine the physiological properties of parvalbumin and its ability to aid in relaxation.  Previous work on the sheepshead assumes that there are differences in the Ca2+ and Mg2+ binding characteristics of the isoforms of parvalbumin.  Although studies have shown differences in the Ca2+ dissociation constant (KD) for parvalbumin from different fish species, there is presently no data on how Mg2+ dissociation rates (the key predictor of the role of parvalbumin in relaxation according to Hou) might vary between species or isoforms of a given species.  To address this issue, we are studying the Ca2+ and Mg2+ binding characteristics of the two parvalbumin isoforms in various fish.  As part of his Senior Thesis, Mohit Sirohi isolated and purified the parvalbumin isoforms from sheepshead and Rainbow Trout using gel filtration and anion-exchange chromatography.  Using his isolation procedure, Ayanah Duhaney isolated parvalbumin from rainbow smelt, and Kristen Sands is working on isolating parvalbumin from Dogfish shark.  Mohit also generated preliminary data indicating differential Ca2+ binding by the two parvalbumin isoforms in Sheepshead.  A new student in my lab, Steven Youseff is working to isolate additional Sheepshead parvalbumin in order to obtain better Ca2+ binding data as well as data to calculate the Mg2+ dissociation constants for the parvalbumin isoforms from sheepshead.  We also hope to collect Ca2+ and Mg2+ binding data for Rainbow Trout this fall.