A Kinetic Characterization of (Na+, K+)-ATPase Activity in the Gills of the Pelagic Seabob Shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae)

We characterize the kinetic properties of a gill (Na+, K+)-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na+, K+)-ATPase activity, but also containing mitochondrial F0F1- and Na+- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na+, K+)-ATPase alpha-subunit with an M-r of a parts per thousand 110 kDa. The alpha-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na+, K+)-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with V-M = 109.5 +/- A 3.2 nmol Pi min(-1) mg(-1) and K-M = 0.03 +/- A 0.003 mmol L-1. Mg2+ (V-M = 109.8 +/- A 2.1 nmol Pi min(-1) mg(-1), K-0.5 = 0.60 +/- A 0.03 mmol L-1), Na+ (V-M = 117.6 +/- A 3.5 nmol Pi min(-1) mg(-1), K-0.5 = 5.36 +/- A 0.14 mmol L-1), K+ (V-M = 112.9 +/- A 1.4 nmol Pi min(-1) mg(-1), K-0.5 = 1.32 +/- A 0.08 mmol L-1), and NH4 (+) (V-M = 200.8 +/- A 7.1 nmol Pi min(-1) mg(-1), K-0.5 = 2.70 +/- A 0.04 mmol L-1) stimulated (Na+, K+)-ATPase activity following site-site interactions. K+ plus NH4 (+) does not synergistically stimulate (Na+, K+)-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K+ binding that can be occupied by NH4 (+), stimulating the enzyme. Ouabain (K-I = 84.0 +/- A 2.1 A mu mol L-1) and orthovanadate (K-I = 0.157 +/- A 0.001 A mu mol L-1) inhibited total ATPase activity by a parts per thousand 50 and a parts per thousand 44 %, respectively. Ouabain inhibition increases a parts per thousand 80 % in the presence of NH4 (+) with a threefold lower K-I, suggesting that NH4 (+) is likely transported as a K+ congener.