A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura)

作者：Farias Daniel L; Lucena Malson N; Garcon Daniela P; Mantelatto Fernando L; McNamara John C; Leone Francisco A^*

来源：Journal of Membrane Biology, 2017, 250(5): 517-534.

DOI：10.1007/s00232-017-9978-6

• Gill (Na+, K+)-ATPase kinetics
• Gill microsomal fraction
• Immunolocalization
• Mangrove crab
• Blue land crab
• Comparative biochemistry

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M (r) of around 110 kDa and hydrolyzes ATP with V (M) = 146.8 +/- 6.3 nmol Pi min(-1) mg protein(-1) and K (M) = 0.05 +/- 0.003 mmol L-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V (M) = 139.4 +/- 6.9 nmol Pi min(-1) mg protein(-1), K (M) = 4.50 +/- 0.22 mmol L-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V (M) = 136.8 +/- 6.5 nmol Pi min(-1) mg protein(-1), K (0.5) = 0.27 +/- 0.04 mmol L-1), K+ (V (M) = 140.2 +/- 7.0 nmol Pi min(-1) mg protein(-1), K (0.5) = 0.17 +/- 0.008 mmol L-1), and NH4 (+) (V (M) = 149.1 +/- 7.4 nmol Pi min(-1) mg protein(-1), K (0.5) = 0.60 +/- 0.03 mmol L-1) shows cooperative kinetics. Ouabain (K (I) = 52.0 +/- 2.6 A mu mol L-1) and orthovanadate (K (I) = 1.0 +/- 0.05 A mu mol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

• 出版日期2017-10