Abstract
Parvalbumin (PV), a soluble protein of skeletal muscle that facilitates muscular relaxation, is expressed across many phyla and has highly conserved Ca2+ binding sites. These features make it ideal for comparisons among organisms that contain highly diverse internal osmolarities. To maintain function in the presence of high internal levels of urea, certain elasmobranch multimeric proteins are stabilized by organic solutes (such as trimethylamine N-oxide, TMAO), which counteract urea. While normal intracellular levels of urea (~400 mM) and TMAO (~200 mM) are not hypothesized to affect calcium-binding protein function in elasmobranch muscle, no studies have determined whether elasmobranch PV is structurally modified to function in the absence of TMAO. This study is the first to address the function of PV in a coastal shark, the Atlantic sharpnose shark (Rhizoprionodon terraenovae). Sequencing of PV from RNA was performed with PCR amplification, cloning, and plasmid sequencing. Parvalbumin from R. terraenovae and a teleost fish, the common carp (Cyprinus carpio) was purified using AMS fractionation, followed by size-exclusion and anion-exchange chromatography. Ca2+ KD of purified shark and carp PV was measured in the presence of a 2:1 ratio of urea/ TMAO and a 2:1 ratio of urea/mannitol by a fluorometric competitive binding assay. Additionally, PV isoform distribution, size, and concentration were determined for three additional coastal sharks by SDS-PAGE, Western blot, and digital comparison to SDS-PAGE standards. R. terraenovae Ca KD averaged 6 nM, and no significant difference for KD was observed between species, or between urea/TMAO and urea/mannitol treatments for both shark and carp. PV titer of the major carp isoform was nine fold higher than the average shark PV titer. It is hypothesized that the monomeric nature of PV may be less susceptible than multimeric proteins to perturbation by urea, as interactions between subunits are less stable than interactions forming protein tertiary structure. The markedly different PV concentrations in the elasmobranch and teleost species used in this study may be indicative of varying properties of fish fast muscle that merit future comparative studies into fast muscle performance.
|
