Some properties of a kidney adenosine triphosphatase relevant to active cation transport

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Abstract
The rate of hydrolysis of ATP to ADP, catalysed by a nuclear fraction from rabbit-kidney-cortex homogenate, has been measured under a variety of conditions designed to find out if the reaction is affected by Na+ and K+ ions, and by ouabain, which are known to affect active ion movements in the intact tissue. The optimum pH within the range 6.5-9.1 for the adenosine-triphosphatase activity in the presence of 110 mM-Na+ ion, 20 mM-K+ ion and equi-molar concentrations (4 mM) of ATP and magnesium chloride was between 7.8 and 8.1. When ouabain (0.33 mM) was also present, the lower activity was much less dependent on pH in this range. Adenosine-triphosphatase activity at pH 8.0 was negligible in the absence of Mg2+ ions and maximal with [Mg2+] [ATP] ratios of 0.5-1-5. The addition of Na+ and K+ ions together, but not separately, stimulated the rate of ATP hydrolysis, the magnitude of the stimulation depending partly on the Na+ and K+ ion concentrations and partly on the ration of these concentrations. With optimum or excess of Na+ ions present, raising the K+ ion concentrations first increased the activity ([K+] :[Na+] ratio less than 1), but further increase in the K+ ion concentration ([K+]:[Na+] ratio greater than 1) caused a fall in activity. With optimum or excess of K+ ions present, the gradual addition of Na+ ions caused an increase in rate, which reached a maximum when the [K+]:[Na+] ratio was less than about 1 and remained constant during further addition of Na+ ions. When the [K+]:[Na+] ratio exceeded about 10, no stimulation of the adenosine-triphosphatase activity was observed. The concentrations of ions required for half-maximal stimulation of the rate of ATP hydrolysis were about 2 mM-K+ ion and about 25 mM-Na+ ion when the sum of the concentrations of these ions was constant at 130 mM. Ouabain (0.1 mM) completely abolished the stimulation caused by Na+ and K+ ions but had no effect on the activity due to Mg2+ ions alone. About 20 [mu]M-ouabain caused half-maximal inhibition of the Na+ ion-plus-K+ ion-dependent adenosine-triphosphatase activity. The inhibition by ouabain was not competitive with respect to ATP. Measurements of the Km gave values in the range 0.4-0.7 mM-ATP for the hydrolysis depending on the presence of Mg2+, Na+ and K+ ions, and 0.6-1.4 mM-ATP for the ouabain-sensitive part of the hydrolysis. In spite of qualifications arising from the heterogeneous nature of the preparation, and from the lack of membrane structural integrity and of directional ion movements in a suspension, the results suggest that a kidney adenosine triphosphatase is stimulated and inhibited in a similar way to active transport and a part of cellular oxygen consumption.