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 +, NH 4 + and ATP of (Na +, K +)-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg 2+, 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 K 0.5 = 63.8 ± 2.9 nmol L − 1 , obeying cooperative kinetics ( n H = 1.9); at the low-affinity sites, hydrolysis obeyed Michaelis–Menten kinetics with K M = 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 ; K M = 7.4 ± 0.4 mmol L − 1 ), Mg 2+ ( V = 132.0 ± 5.3 U mg − 1 ; K 0.5 = 0.36 ± 0.02 mmol L − 1 ), NH 4 + ( V = 245.6 ± 9.8 U mg − 1 ; K M = 4.5 ± 0.2 mmol L − 1 ) and K + ( V = 140.0 ± 4.9 U mg − 1 ; K M = 1.5 ± 0.1 mmol L − 1 ) followed a single saturation curve and, except for Mg 2+, obeyed Michaelis–Menten kinetics. Under optimal ionic conditions, but in the absence of NH 4 +, ouabain ( K I = 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 F 0F 1, V- and P-ATPases, but not Na +-, K +- or Ca 2+-ATPases as contaminants in the gill microsomal preparation. (Na +, K +)-ATPase activity was synergistically modulated by NH 4 + and K +. At 20 mmol L − 1 K +, a maximum rate of V = 290.8 ± 14.5 U mg − 1 was seen as NH 4 + concentration was increased up to 50 mmol L − 1 . However, at fixed NH 4 + 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 NH 4 +, 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 NH 4 +, as yet undescribed for other (Na +, K +)-ATPases, and should provide a better understanding of NH 4 + excretion in pagurid crabs.