CHARACTERIZATION OF ESSENTIAL ARGINYL RESIDUES IN SHEEP KIDNEY (NA+ + K+)-ATPASE

Treatment of highly purified sheep kidney medulla (Na+ + K+)-ATPase with 2,3-butanedione results in a rapid inactivation of the enzyme. Contrary to a previous report using rabbit kidney enzyme (DePont et al., Biochim. Biophys. Acta (1977) 482, 213), the inactivation is biphasic under a variety of experimental conditions, with a rapid, initial inactivation which is followed by a slower loss of activity. The second, slower phase of the inhibition obeys pseudo-first order kinetics, with a second order rate constant for inhibition of 20 min-1M-1. ATP and ADP provide no protection in the initial phase of the inhibition, but protect the enzyme completely from the second phase of the inhibition. AMP, while less effective than ATP and ADP, provides a partial protection of the enzyme activity from inhibition by 2,3-butanedione. Inorganic phosphate provides partial protection in both phases of the inactivation. Adenosine alone is without effect, but adenosine plus inorganic phosphate provides a greater protection than phosphate alone. The results indicate that either (1) two or more active site residues or (2) a single arginine, experiencing different reactivities in two different active site conformations, are essential to (Na+ + K+)-ATPase activity. © 1979.