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

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 alpha-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single alpha-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 +/- A 2.3 U mg(-1), K (0.5) = 0.10 +/- A 0.01 mmol L-1. Stimulation by Na+ (V (M) = 110.0 +/- A 3.3 U mg(-1), K (0.5) = 1.30 +/- A 0.03 mmol L-1), Mg2+ (V (M) = 115.0 +/- A 4.6 U mg(-1), K (0.5) = 0.96 +/- A 0.03 mmol L-1), NH4 (+) (V (M) = 141.0 +/- A 5.6 U mg(-1), K (0.5) = 1.90 +/- A 0.04 mmol L-1), and K+ (V (M) = 120.0 +/- A 2.4 U mg(-1), K (M) = 2.74 +/- A 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 +/- A 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 (a parts per thousand 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.