Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica)

We have compared the primary sequence and enzymatic properties of the sarco plasmic reticulum Ca2+-ATPases from a cold-tolerant frog Rana sylvatica wit h those of a closely related cold-intolerant frog, Rana clamitans, Sarcopla smic reticulum isolated from leg muscles of both species contains a major p rotein (similar to 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca2+-ATPase type 1 (SERCA1). The apparent mole cular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs d o not indicate a difference in molecular weight, thus suggesting post-trans lational protein modification of R. sylvatica SERCA1, Comparison of the tem perature dependence of both ATP hydrolysis and Ca2+ transport indicates tha t R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degreesC and an similar to2-fold greater Ca2+-ATPase activity near 0 deg reesC. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten ki netics with ATP and Ca2+ as opposed to the two-site ATP kinetics and positi ve cooperativity with Ca2+ observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, an d this property may be altered in R. sylvatica SERCA1. Primary sequence com parison shows that R. sylvatica SERCA1 exhibits seven unique amino acid sub stitutions, three of which are in the ATP binding domain. We also report fo r the first time the presence of alternative splicing in the frog, resultin g in isoforms SERCA1a and SERCA1b, Thus, it appears that the low temperatur e muscle contractility of R. sylvatica can be explained partially by signif icant functional and structural differences in SERCA1.