The kinetic properties of a microsomal gill (Na+, K+)-ATPase from juvenile and adult Amazon River shrimp Macrobrachium amazonicum were characterized using the synthetic substrate p-nitrophenylphosphate. The substrate was hydrolyzed revealing cooperative kinetics at maximum rates of 71.3 ± 1.1 U mg-1 and 82.7 ± 2.3 U mg-1 with K0.5 = 1.45 ± 0.02 mmol L-1 and 1.52 ± 0.04 mmol L-1 for juveniles and adults, respectively. Stimulation of K+-phosphatase activity by Mg2+ and K+ also exhibited cooperative kinetics independently of ontogenetic stage. While the K0.5 values estimated for Mg2+ and K+ stimulation of K+-phosphatase activity were very similar, maximum rates for adult shrimps were ≈1.8-fold greater than that for juveniles. NH4+ stimulation resulted in a ≈10-fold increase in K0.5 for both stages compared to Mg2+ and K+. Further, when stimulated by NH4+ plus K+, p-nitrophenylphosphate hydrolysis was mostly unchanged, suggesting that NH4+ and K+ bind to the same site. While K0.5 values varied, stimulation by NH4+ plus K+ was not synergistic. Increasing NH4+ or K+ concentrations do not displace the corresponding ion from the cation-binding site. Ouabain partially inhibited p-nitrophenylphosphatase activity (KI = 142.0 ± 5.6 µmol L-1 and 156.0 ± 6.2 µmol L-1 for juveniles and adults, respectively); however, inhibition was more effective when NH4+ was present (KI = 117.1 ± 4.6 µmol L-1 and 47.2 ± 1.9 µmol L-1 for juveniles and adults, respectively). Although the activity of ATPases other than the (Na+, K+)-ATPase are considerably greater in adult shrimps, the kinetic properties of the gill K+-phosphatase activity in the two life cycle stages, and the affinities for p-nitrophenylphosphate and ions are similar.
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