The mechanism of the Na+/K+-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K+-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 μg/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 μg/ml. Na+/K+-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na+-ATPase activity was stimulated to a greater extent (up to 350%), whereas K+-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K+-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K+-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K+-or Na+-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K+-ATPase at site(s) located on the extracellular moiety of the enzyme.
|Number of pages||7|
|Journal||Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular|
|Publication status||Published - May 31 1990|
- ATPase, Na/K-
ASJC Scopus subject areas
- Molecular Biology
- Structural Biology