“Ouabain-insentive Na+ATPase activities in muscle of Chasmagnathus granulatus upon hyperegulation”

PINONI, S.; LOPEZ MAÑANES A.A.

Buenos Aires

Congreso; VII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología; 2006

Two Na+-stimulated ATPases, an ouabain–sensitive Na+K+ATPase (NKA) and an ouabain–insensitive Na+ATPase (NA), with distinct properties, appear to occur in several animal tissues. In invertebrates, NA is involved in intracellular homeostasis of Na+. As part of our studies on regulatory mechanisms at the biochemical level of the euryhaline crab C. granulatus from Mar Chiquita coastal lagoon (Bs. As. Province), we previously showed the occurrence of salinity dependent NKA and alkaline phosphatase activities in chela muscle  suggesting a role for this tissue in regulatory mechanisms evoked upon osmo-ionic stress. The aim of this work was to study the occurrence and biochemical characteristics of NA in chela muscle. Male adult crabs were acclimated for at least 10 days to 10‰ salinity. NA activity was determined in chela muscle homogenates (0.25M Sucrose/0.5mM EGTA-Tris pH 7.4) by measuring ATP hydrolysis (0.05-13.00 mM) in the presence of (mM): 20 imidazole (pH 6.2-7.8)/130 NaCl/0.5 EGTA/1 ouabain/1 NaN3, in the presence (furosemide-insensitive, FI, NA activity) or the absence of  2 furosemide. The difference of both assays represents the furosemide-sensitive (FS) NA activity. Control: without NaCl. 3-6 independent experiments were carried out. Two NA activities were detected in chela muscle, a FI and a FS (88.2±11.9 and 117.5±23.2 nmoles Pixmin-1xmg prot-1 respectively). FI and FS activities showed a Michaelis-Menten kinetics (Km= 0.021 and 0.224 mM, respectively). Maximal furosemide inhibition was at 2 mM (I50=1.4 mM). Maximal activities occurred at pH 7.4 and NaCl 130 mM. The possible role of NA activities in mechanisms of adjustments secondary to hyperegulation in muscle of C. granulatus remains to be investigated.