Modulation by ammonium ions of gill microsomal (Na +,K +)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion

Abstract

The modulation by Na +, K +, NH 4 + and ATP of the (Na +,K +)-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4±2.1 U mg −1 and K 0.5=54.0±3.6 nM, obeying cooperative kinetics ( n H=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis–Menten kinetics with K M=55.0±3.0 μM and V=271.5±17.2 U mg −1. This is the first demonstration of a crustacean (Na +,K +)-ATPase with two ATP hydrolyzing sites. Stimulation by sodium ( K 0.5=5.80±0.30 mM), magnesium ( K 0.5=0.48±0.02 mM) and potassium ions ( K 0.5=1.61±0.06 mM) exhibited site–site interactions, while that by ammonium ions obeyed Michaelis–Menten kinetics ( K M=4.61±0.27 mM). Ouabain ( K I=147.2±7.2 μM) and orthovanadate ( K I=11.2±0.6 μM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na +,K +)-ATPase activity by ammonium ions, and the excretion of NH 4 + in benthic crabs are discussed.