Meeting Abstract

P1.85  Jan. 4  Modification of the α-subunit may modulate Na⁺,K⁺-ATPase activity in gills of the euryhaline crabs Callinectes sapidus and Carcinus maenas MCKEON-FISH, A.F.**; TREITLER, P.C.; LOVETT, D.L.; The College of New Jersey, Ewing lovett@tcnj.edu

Estuarine crabs hyperregulate after transfer from high to low salinity. Even though crabs maintain a stable hemolymph osmolality shortly after transfer, Na⁺,K⁺-ATPase (ATPase) activity and the amount of ATPase protein in posterior gills do not change during initial acute exposure to dilute seawater. Western blots of posterior gill homogenates yield two distinct (113 and 131 kDa) forms of the α-subunit of ATPase when treated with anti-α-subunit. Crabs acclimated to dilute seawater have larger amounts of the 131 kDa form. To infer whether glycosylation or phosphorylation of the α-subunit may modulate activity, we have examined whether either process accounts for the difference between the two electrophoretic forms. Western blots of gill homogenates treated to detect glycoproteins yielded only the 131 kDa band. When homogenates were deglycosylated with trifluoromethanesulfonic acid, Western blots treated with anti-α-subunit yielded only the 113 kDA band, while blots treated to detect glycoproteins yielded no bands. Thus, it appears that the 113 kDA form of the α-subunit is glycosylated to produce the 131 kDA form. In contrast, when homogenates were dephosphorylated with calf intestinal phosphatase, Western blots yielded both α-subunit bands. The relative proportion of each form in dephosphorylated homogenates was similar to that in homogenates treated with phosphatase inhibitors. Although others have shown that dopamine and cAMP stimulate ATPase activity in crab gills (suggesting that activity is modulated through phosphorylation of the enzyme), it appears that glycosylation of the enzyme may also be involved in modulating ATPase activity during acute change in seawater salinity. (Supported in part by NSF grant IBN-0240903).