A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na+, K+, NH4+ and ATP of (Na+, K+)-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg2+, Na+ and K+ concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V = 19.1 ± 0.8 U mg− 1 and K0.5 = 63.8 ± 2.9 nmol L− 1, obeying cooperative kinetics (nH = 1.9); at the low-affinity sites, hydrolysis obeyed Michaelis–Menten kinetics with KM = 44.1 ± 2.6 μmol L− 1 and V = 123.5 ± 6.1 U mg− 1. Stimulation by Na+ (V = 149.0 ± 7.4 U mg− 1; KM = 7.4 ± 0.4 mmol L− 1), Mg2+ (V = 132.0 ± 5.3 U mg− 1; K0.5 = 0.36 ± 0.02 mmol L− 1), NH4+ (V = 245.6 ± 9.8 U mg− 1; KM = 4.5 ± 0.2 mmol L− 1) and K+ (V = 140.0 ± 4.9 U mg− 1; KM = 1.5 ± 0.1 mmol L− 1) followed a single saturation curve and, except for Mg2+, obeyed Michaelis–Menten kinetics. Under optimal ionic conditions, but in the absence of NH4+, ouabain (KI = 117.3 ± 3.5 μmol L− 1) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F0F1, V- and P-ATPases, but not Na+-, K+- or Ca2+-ATPases as contaminants in the gill microsomal preparation. (Na+, K+)-ATPase activity was synergistically modulated by NH4+ and K+. At 20 mmol L− 1 K+, a maximum rate of V = 290.8 ± 14.5 U mg− 1 was seen as NH4+ concentration was increased up to 50 mmol L− 1. However, at fixed NH4+ concentrations, no additional stimulation was found for increasing K+ concentrations (V = 135.2 ± 4.1 U mg− 1 and V = 236.6 ± 9.5 U mg− 1 and for 10 and 30 mmol L− 1 NH4+, respectively). This is the first report to detail ionic modulation of gill (Na+, K+)-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K+ and NH4+, as yet undescribed for other (Na+, K+)-ATPases, and should provide a better understanding of NH4+ excretion in pagurid crabs.