Subcellular Localization and Kinetic Characterization of a Gill (Na⁺, K⁺)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

Abstract

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg⁻¹, K _0.5 = 0.10 ± 0.01 mmol L⁻¹. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg⁻¹, K _0.5 = 1.30 ± 0.03 mmol L⁻¹), Mg²⁺ (V _M = 115.0 ± 4.6 U mg⁻¹, K _0.5 = 0.96 ± 0.03 mmol L⁻¹), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg⁻¹, K _0.5 = 1.90 ± 0.04 mmol L⁻¹), and K⁺ (V _M = 120.0 ± 2.4 U mg⁻¹, K _M = 2.74 ± 0.08 mmol L⁻¹) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L⁻¹. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.