To better comprehend the adaptive strategies that have led to the invasion of the freshwater biotope by hyperegulating Crustacea, we have employed a microsomal, gill tissue fraction to kinetically characterize the (Na⁺, K⁺)-ATPase in several portunid and diogenid crabs, and palaemonid shrimps, that inhabit different, variable salinity regimes, including marine, estuarine and fresh waters, during their life cycles.

Our studies, employing both fresh-caught and low salinity-acclimated specimens, have disclosed various important findings. The (Na⁺, K⁺)-ATPase from the euryhaline, coastal swimming crab Callinectes danae possesses two distinct ATP binding sites: high-affinity sites showing a specific activity of 35.4 nmol Pi/min/mg and K_0.5= 54.0 nM; and low-affinity sites exhibiting a specific activity of 271.5 nmol Pi/min/mg and K_M= 55.0 µM. This is the first demonstration of a crustacean (Na⁺, K⁺)-ATPase possessing two different sites for ATP hydrolysis.

We have also shown that ammonium and potassium ions synergistically stimulate ATP hydrolysis by the enzyme from C. danae, increasing specific activity up to 90%, which suggests the binding of these ions to different sites. This is also the first demonstration of synergistic stimulation of the (Na⁺, K⁺)-ATPase by potassium and ammonium ions. ATP hydrolysis by the enzyme from the euryhaline, hyperosmoregulating, palaemonid shrimp, Macrobrachium olfersii, is also synergistically stimulated (≈25%) by ammonium ions at saturating potassium ion concentrations. However, potassium ions do not stimulate activity under saturating ammonium ion concentrations. Given that crustaceans are ammoniotelic, and since most ammonia is excreted across the gill epithelium, the synergistic stimulation of the gill (Na⁺, K⁺)-ATPase by potassium and ammonium ions appears to be physiologically relevant. Callinectes danae is a benthic crab that buries in shallow water sediments which may contain high ammonia concentrations; while buried, continuous ammonia excretion may contribute to further local concentration increases. Thus, marine and estuarine benthic crabs in particular, may encounter ambient ammonia concentrations that exceed hemolymph titers, resulting in ammonia influx as a consequence of their elevated gill epithelial permeability, which may have led to the evolutionary selection of a mechanism for NH₄⁺ excretion against its gradient, and in which the (Na⁺, K⁺)-ATPase appears to be directly involved by transporting NH₄⁺ from the hemolymph. In Macrobrachium olfersii, our findings suggest that at high NH₄⁺ ion concentrations, the (Na⁺, K⁺)-ATPase exposes a new binding site, and subsequent NH₄⁺ ion binding stimulates ATP hydrolysis to rates higher than those for K⁺ ions alone, a putative, fine-tuning mechanism of physiological importance to osmoregulatory and excretory processes in palaemonid shrimps.

 We have also investigated the kinetic properties of these gill (Na⁺, K⁺)-ATPases using a synthetic, organic substrate, p-nitrophenylphosphate (PNPP). Our findings show that while ATP and PNPP are hydrolyzed at a common site, K⁺-phosphatase activity is not synergistically stimulated by potassium and ammonium ions. These studies also reveal that the usefulness of PNPP as a substrate to characterize K⁺-phosphatase activity in comparative osmoregulatory studies involving (Na⁺, K⁺)-ATPase activities in crustacean gill tissues, and for consistent comparison with well known mechanistic properties of the vertebrate enzyme.