ALBERT

Notizen: Summary Basal and trypsin-stimulated adenosine triphosphatase activities ofEscherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5mm, Vmax (minus trypsin) = 1.6µmol·min−1·mg protein−1, Vmax (plus trypsin) = 2.44µmol·min−1·mg protein−1; for the soluble ATPase: [S0.5] = 1.2mm, Vmax (-trypsin) = 4µmol·min−1·mg protein−1; Vmax (+trypsin) = 6.6µmol·min−1·mg protein−1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4mm) showed two sites (groups) with different K ms: at low ATP the values were 0.38 and 1.4mm for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20mm, respectively. Mg2+ saturation at constant ATP (8mm) revealed michaelian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulatesE. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors ofEscherichia coli ATPase. The Ki values for Pi were 1.6 ± 0.1mm for the membrane-bound ATPase and 1.3 ± 0.1mm for the enzyme in soluble form, the Ki values for ADP being 1.7mm and 0.75mm for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4mm) inhibited the membrane-bound enzyme by 60–70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20µ m) inhibited both states ofE. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.