K+-independent ouabain-insensitive Na+-ATPase activity in hepatopancreas of Neohelice granulata

MENDEZ, E.; LÓPEZ MAÑANES, A,A.

Encuentro; XVI Annual Meeting Argentine Biology Society; 2014

A K+-independent ouabain?insensitive Na+ATPase (NA), with distinct properties, occur in several mammals tissues. In comparison information in invertebrates are scarce. We have shown the occurrence of K+-independent, ouabain-insensitive, furosemide-sensitive Na+-ATPase activity (NA) in muscle of the euryhaline crab N. granulata. Studies on NA in hepatopancreas (HP) of euryhaline crabs are lacking. The aim of this work was to study the occurrence and biochemical characteristics of NA in HP. Adult male crabs were maintained for at least 10 days at 35? salinity. NA was determined in HP homogenates (0.25M Sucrose/0.5mM EGTA-Tris pH 7.4). NA (μmol Pix min-1x mgprot-1) was assayed by hydrolysis of ATP in a reaction mixture containing (mM): 10 MgCl2, 0.5 EGTA, 1 ouabain, 1 Na3N, in 20 imidazole buffer, pH 7.4, without or with of 2mM furosemide, 30ºC (Na+ curve: 0-150 mM; ATP 15 mM) (ATP curve: 0.5-50; 100 mM NaCl) (time curve: 10-30 min; 100 mM NaCl, 15 mM ATP) (furosemide curve: 0.1-2 mM; 100 mM NaCl, 15 mM ATP). NA was estimated as the difference between assays with or without 2mM furosemide. Maximal activities occurred at NaCl 100 mM, 15 mM ATP and for 20 minutes of incubation. Maximal inhibition of NA was found at 2 mM. The results show the occurrence for the first time, of NA in HP of an euryhaline crab