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Kinetic and molecular characterization of gill tissue (Na+,K+)-ATPase from Cardisoma guanhumi (Latreille)crab

Grant number: 13/22605-1
Support Opportunities:Regular Research Grants
Duration: July 01, 2014 - June 30, 2016
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Francisco de Assis Leone
Grantee:Francisco de Assis Leone
Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated researchers: Carlos Frederico Leite Fontes ; Daniela Pereira Garçon ; Fernando Luis Medina Mantelatto ; John Campbell McNamara ; Marcelo Rodrigues Pinto


This project is a continuation of our research line, that has been supported by FAPESP (2013/00976-8; 2013/24252-9; 2010/17534-0; 2010/16115-3; 2010/06395-9; 2006/56435-1; 2006/56759-1; 2003/01233-7) aiming the systematic study of the kinetic structural properties of (Na,K)-ATPase from gill tissue of different species of crabs and shrimps in order to establish a model for the adaptation of crustaceans to environments of different salinities. Crustacean hemolymph osmolality is maintained by the (Na,K)-ATPase and the adaptation mechanism suggests that the responses of crustaceans must involve a rapid activation of ion transport that must be primary to any other physiological responses of these animals. The current project aims to investigate the cellular signaling linked to the regulation of gill (Na,K)-ATPase of Cardisoma guanhumi and its direct consequences on the uptake of NaCl during acclimation to hyposaline (salinity below 330 salinity) and marine (around 330 salinity) environments. The following studies will be undertaken: 1) preparation of gill (Na,K)-ATPase from Cardisoma guanhumi. 2) kinetic characterization of (Na,K)-ATPase modulation by ATP, PNPP, sodium, potassium, magnesium, ammonium and ouabain. 3) expression of the gill Na,K-ATPase. 4) subcellular localization of (Na,K)-ATPase. 5) to quantify activity of enzymes involved in oxidative stress in animals acclimated to different salinities. 6) to study the phosphoenzyme formation involving enzyme phosphorylation under adequate modulator concentrations; polyamine effects on phosphoenzyme stability; polyamine effects on enzyme dephosphorylation in the presence of K+; western blot analysis of FXYD2 after phosphorylation by PKA and PKC; preparation and extraction of phosphorylated FXYD2. 7) to study of the effect of endogenous and exogenous FXYD2 on (Na,K)-ATPase activity. 8) to study the effect of polyamines on (Na,K)-ATPase activity. 9) elaboration of an experimental protocol for gill cell culture. 10) to correlate the FXYD2 with protein kinases stimulated by polyamines and its final action on the activity of Na,K-ATPase in different saline media, using cultured gill cells. 11) analysis of Na,K-ATPase activity of C. guanhumi acclimated to different conditions (salinity, ammonium, nitrite, heavy metals). (AU)

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Scientific publications (5)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
FARIAS, DANIEL L.; LUCENA, MALSON N.; GARCON, DANIELA P.; MANTELATTO, FERNANDO L.; MCNAMARA, JOHN C.; LEONE, FRANCISCO A.. A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura). Journal of Membrane Biology, v. 250, n. 5, p. 517-534, . (13/24252-9, 13/22605-1, 13/00976-8, 10/17534-0, 10/50188-8)
LEONE, FRANCISCO A.; GARCON, DANIELA P.; LUCENA, MALSON N.; FALEIROS, ROGERIO O.; AZEVEDO, SERGIO V.; PINTO, MARCELO R.; MCNAMARA, JOHN C.. Gill-specific (Na+, K+)-ATPase activity and alpha-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura). COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, v. 186, p. 59-67, . (13/24252-9, 11/15776-9, 10/06395-9, 13/22605-1, 07/04859-5, 10/17534-0)
ANTUNES, CLAUDIA DOI; LUCENA, MALSON NEILSON; GARCON, DANIELA PEREIRA; LEONE, FRANCISCO ASSIS; MCNAMARA, JOHN CAMPBELL. Low salinity-induced alterations in epithelial ultrastructure, Na+/K+-ATPase immunolocalization and enzyme kinetic characteristics in the gills of the thinstripe hermit crab, Clibanarius vittatus (Anomura, Diogenidae). JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL AND INTEGRATIVE PHYSIOLOGY, v. 327, n. 6, p. 380-397, . (13/22605-1, 10/17534-0, 13/24252-9, 10/50188-8)
PINTO, MARCELO R.; LUCENA, MALSON N.; FALEIROS, ROGERIO OLIVEIRA; ALMEIDA, EDUARDO ALVES; MCNAMARA, JOHN C.; LEONE, FRANCISCO A.. Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae). AQUATIC TOXICOLOGY, v. 170, p. 13-23, . (13/22605-1, 10/17534-0, 11/15776-9, 13/24252-9)

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