On the mechanism of stimulation of the Na/K pump of LK sheep erythrocytes by anti-L antibody.

Abstract

Studies were undertaken to explore the mechanism of stimulation of the Na/K pump in LK sheep erythrocytes by anti-L antibody. First, the numbers of functioning pump sites were determined by correlating [3H]ouabain binding with levels of inhibition of the pump. Untreated (control) cells had approximately 41 pumps per cell, and anti-L treatment caused an increase in the number of functioning pumps to approximately 85 per cell. Reducing the intracellular K concentration, [K]c, to near zero caused an increase in the number of pumps in control cells, but not in anti-L cells, such that the numbers of pumps per cell were about the same in the two cell types. These results led to the prediction that Kc is a noncompetitive inhibitor of the pump in control cells, and that anti-L stimulates the pump and increases number of functioning pumps by reducing noncompetitive inhibition by Kc. Kinetic studies were undertaken to test this prediction: activation of the pump by increasing [Na]c was measured at three fixed levels of [K]c. In control cells, the apparent maximum velocity of the pump (J'max) was reduced approximately threefold by raising [K]c from 0.2 to 9 mmol/liter cells, demonstrating noncompetitive inhibition by Kc. In anti-L cells, J'max did not vary with [K]c, which shows that, as predicted, anti-L abolishes the noncompetitive inhibition by Kc. The modification of the kinetic properties of the pumps by the antibody is highly specific in that affinities for Nac and Ko as substrates are unaffected. However, the effect of the antibody on noncompetitive inhibition by Kc does not explain the stimulation of the pump fully since there is significant stimulation at near-zero [K]c.