Kinetics of the inhibition of the Na-K pump by tetrapropylammonium chloride.

1. The tetrapropylammonium ion (TPA) acts as a mixed-type (with K) inhibitor of the Na-K pump. The kinetics of the process suggest that combination of the pump with a single TPA ion is sufficient for inhibition. 2. TPA inhibits the partial reactions of the Na-K pump (the uncoupled Na outflux, the Na-Na exchange, and K-K exchange). 3. TPA inhibits ouabain binding to the pump and this inhibitory effect is enhanced by external Na. The inhibitory effect of TPA on the pump rate is also promoted by external Na. 4. A Lineweaver-Burk plot of the reciprocal of the ouabain-sensitive K influx versus the reciprocal of the external K concentration is approximately a straight line if the measurements are made in Na-free solutions. TPA increases the apparent Michaelis constant (K 1/2) for K and the plot remains straight. 5. The Lineweaver-Burk plot is parabolic when the measurements are made in solutions which contain Na. TPA both increases the apparent K 1/2 for K and makes the curve more parabolic. 6. The characteristics of pump inhibition by TPA are similar to those for strophanthidin. In both cases the kinetic behaviour is consistent with a model in which the inhibitor binds: with greatest affinity to the pump form free of K; with less affinity to the pump form with a single bound K; and with least affinity to the pump form with two bound K.

Interaction of HK and LK goat red blood cells with ouabain.

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (K(c)) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low K(c) cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal K(c), it does not alter the apparent number of sites in LK cells when K(c) has been reduced.

Antibody-induced alterations in the kinetic characteristics of the Na:K pump in goat red blood cells.

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K(c)), increasing K(c) at first stimulates the active K influx in HK cells, but at higher K(c) the pump is inhibited. These results suggest that in HK cells K(c) acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na(c)) at the Na translocation sites. In LK cells, K(c) inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K(c). The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K(c) LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.