The kinetic properties of the substrate and cation-binding sites of a microsomal gill (Na+, K+)-ATPase from the hermit crab Clibanarius vittatus were characterized using the synthetic substrate p-nitrophenylphosphate. Substrate was hydrolyzed obeying Michaelis-Menten kinetics at a maximum rate of 47.4 ± 1.9 U mg-1 with KM = 1.0 ± 0.04 mmol L-1. Stimulation of K+-phosphatase activity by Mg2+ (VM = 43.9 ± 1.5 U mg-1 and K0.5 = 0.6 ± 0.1 mmol L-1), K+ (VM = 48.9 ± 2.4 U mg-1 and K0.5 = 3.7 ± 0.1 mmol L-1) and NH4+ (VM = 50.1 ± 1.7 U mg-1 and K0.5 = 19.3 ± 0.7 mmol L-1) showed cooperative kinetics. Stimulation by K+ or NH4+ of K+-phosphatase activity was similar, enzyme specific activity not varying noticeably in the presence of both ions. The data suggest that after binding of either K+ or NH4+ to their sites, increasing concentrations of the other do not displace the first ion from its binding site. Na+ (KI = 16.1 ± 0.2 mmol L-1), ouabain (KI = 340.4 ± 16.1 µmol L-1) and orthovanadate inhibited 70-90% of p-nitrophenylphosphatase activity. This is the first known kinetic characterization of K+-phosphatase activity in a hermit crab and provides a useful tool for comparative biochemical studies of (Na+, K+)-ATPase activities in crustacean gill tissues.
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