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 F 0 F 1 - and Na + - and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na + , K + )-ATPase α-subunit with an M r of ≈110 kDa. The α-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 ± 3.2 nmol Pi min −1 mg −1 and K M = 0.03 ± 0.003 mmol L −1 . Mg 2+ (V M = 109.8 ± 2.1 nmol Pi min −1 mg −1 , K 0.5 = 0.60 ± 0.03 mmol L −1 ), Na + (V M = 117.6 ± 3.5 nmol Pi min −1 mg −1 , K 0.5 = 5.36 ± 0.14 mmol L −1 ), K + (V M = 112.9 ± 1.4 nmol Pi min −1 mg −1 , K 0.5 = 1.32 ± 0.08 mmol L −1 ), and NH 4 + (V M = 200.8 ± 7.1 nmol Pi min −1 mg −1 , K 0.5 = 2.70 ± 0.04 mmol L −1 ) stimulated (Na + , K + )-ATPase activity following site–site interactions. K + plus NH 4 + 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 NH 4 + , stimulating the enzyme. Ouabain (K I = 84.0 ± 2.1 µmol L −1 ) and orthovanadate (K I = 0.157 ± 0.001 µmol L −1 ) inhibited total ATPase activity by ≈50 and ≈44 %, respectively. Ouabain inhibition increases ≈80 % in the presence of NH 4 + with a threefold lower K I , suggesting that NH 4 + is likely transported as a K + congener.