Factors influencing the onset of ouabain inhibition of Na,K-ATPase from guinea-pig myocardium

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Abstract
1 The onset of ouabain inhibition was quantified by analysis with an integrated rate equation from experiments in which the activity of Na,K-ATPase from guinea-pig myocardium had been altered with adenosine 5′-triphosphate (ATP, 0.3–9 mmoll−1) in the absence and presence of a detergent. 2 Under control conditions with increasing ouabain (0.1–100 μmoll−1) and ATP (0.3–1 mmoll−1) concentrations, inhibition developed faster. The acceleration by ouabain became less effective at saturating concentrations leading to a non-linear relationship between pseudo-first-order rate constants of inhibition and ouabain concentration. With a rise of ATP to 3 and 9 mmoll−1, i.e., near total Mg concentration (5 mmoll−1), inhibition was retarded presumably because the free concentrations of Mg and uncomplexed ATP changed. Varying the ATP concentration had little effect on ouabain potency at steady state; Hill coefficients were < 1. 3 The detergent alamethicin (23 μg ml−1) neither interfered with Na,K-ATPase activity nor with inhibition at steady state but accelerated its onset. This supports a role for a lipid barrier in the development of inhibition. 4 While the reaction of low concentrations of ouabain with the receptors seemed to govern inhibition rate, with an increase in steroid concentration in the presence of alamethicin, ATP-dependent enzyme activity interfered with the onset of inhibition. The transition of the enzyme between ouabain-sensitive and ATP-hydrolytic conformations consequently causes the non-linear concentration-dependence of pseudo-first-order rate constants. As the Hill coefficient was < 1, a reaction of ouabain with two receptors also could have contributed to the special concentration-dependence of inhibition rates.

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