rd
23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
th
th
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
(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
immunolocalization.
guanhumi,
kinetic
characterization,
Supported by: FAPESP, CNPq, FAPEAM/ INCT-ADAPTA
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)
(Na+,K+)-ATPase,
[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.