23 Congress of the International Union for Biochemistry and Molecular Biology
44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
Immunolocalization and Kinetic Characterization of a (Na+, K+)-ATPase in the Gills
of the Blue Land Crab Cardisoma guanhumi (Latreille, 1825)
Farias, D.L.1; Lucena, M.N.1; McNamara, J.C.2; Pinto, M.R.1; Leone, F.A.1
Departamento de Química e (2)Departamento de Biologia, FFCLRP/ Universidade de
São Paulo, São Paulo, Brasil.
INTRODUCTION: The (Na+,K+)-ATPase is an integral plasma membrane protein that
provides the driving force sustaining many ion-transport systems. The enzyme couples
the exchange of 3 intracellular Na+ and 2 extracellular K+ with the hydrolysis of a single
ATP molecule, and is subject to complex regulation. Crustacean posterior gills show
high levels of (Na+,K+)-ATPase activity and constitute the primary site of ion uptake from
their ambient medium to the hemolymph. OBJECTIVES: To provide a kinetic
characterization of the (Na+,K+)-ATPase from the posterior gill tissue of C. guanhumi,
and to obtain information about its physiological and biochemical adaptation.
MATERIALS AND METHODS: Enzyme immunolocalization was performed using
confocal and fluorescence microscopy. Total ATPase activity was assayed at 25 °C
using a Pyruvate kinase/Lactic dehydrogenase coupling system in which ATP hydrolysis
was coupled to NADH oxidation at 340 nm. RESULTS AND DISCUSSION: Western
blotting with monoclonal antibodies revealed a single ≈110-kDa band, corresponding to
the (Na+,K+)-ATPase -subunit. The -subunit is distributed predominantly in the
basolateral region of the gill pillar cells. The enzyme hydrolyzed ATP with a specific
activity of 148.46 nmol min-1 mg-1 and KM = 57.6 µmol L-1 obeying Michaelian kinetics.
Magnesium stimulation (K0.5 = 0.32 mmol L-1) was cooperative; sodium stimulation (KM =
4.5 mmol L-1) obeyed Michaelian kinetics. Total ATPase activity was inhibited 80% by
ouabain (KI of 51.9 ± 2.59 µmol L-1). CONCLUSION: This is the first kinetic
characterization of a microsomal (Na+,K+)-ATPase from the posterior gill tissue of C.
guanhumi demonstrates that this enzyme is a significant regulator during the
development of this species.
Keywords: Cardisoma
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)
[jcm1] Comentário: Essa conclusão
não decorre dos dados apresentados. Não há
nada sobre diferentes fases de
desenvolvimento? Não entendi bem aqui.
Também, não há nada sobre NH4+ nos
resultdaos? Tirar dos objetivos?
Cuidado “Blue crab” (=Callinectes,
marinho) e “Blue land crab” (=Cardisoma,
terrestre) são muito diferentes.