Pre-steady-state kinetic study of the mechanism of inhibition of the plasma membrane Ca(2+)-ATPase by lanthanum.

Lanthanides are known to be effective inhibitors of the PMCa(2+)-ATPase. The effects of LaCl3 on the partial reactions that take place during ATP hydrolysis by the calcium-dependent ATPase from plasma membrane (PMCa(2+)-ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. LaCl3 added before phosphorylation (K0.5 = 2.8 +/- 0.2 microM) raised the kapp of the E2-->E1 transition from 14 +/- 2 to 23 +/- 4 s-1. The effect was independent of Ca2+ and Mg2+, as if La3+ substituted for Mg2+ and/or Ca2+ in accelerating the formation of E1 with higher efficiency. At non-limiting conditions, LaCl3 doubled the apparent concentration of E1 in the enzyme at rest with Ca2+ and Mg2+. LaCl3 during phosphorylation (K0.5 near 20 microM) lowered the vo of the reaction from 300 +/- 20 to 60 +/- 7 pmol/mg of protein/s, a close rate to that in the absence of Mg2+. This effect was reversed by Mg2+ (and not by Ca2+), and the K0.5 for Mg2+ as activator of the phosphorylation reaction increased linearly with the concentration of LaCl3, suggesting that La3+ slowed phosphorylation by displacing Mg2+ from the activation site(s). If added before phosphorylation, LaCl3 lowered the kapp for decomposition of EP to 0.8 +/- 0.1 s-1, a value which is characteristic of phosphoenzyme without Mg2+. The K0.5 for this effect was 0.9 +/- 0.5 microM LaCl3 and increased linearly with the concentration of Mg2+. If added after phosphorylation, LaCl3 did not change the kapp of 90 +/- 7 s-1 of decomposition of EP, suggesting that La3+ displaced Mg2+ from the site whose occupation accelerates the shifting of E1P to E2P. In medium with 0.5 mM MgCl2, 2 microM LaCl3 lowered rapidly the rate of steady-state hydrolysis of ATP by the PMCa(2+)-ATPase to a value close to the rate of decomposition of EP made in medium with LaCl3. Increasing MgCl2 to 10 mM protected the PMCa(2+)-ATPase against inhibition during the first 10 min of incubation. Results show that combination of La3+ to the Mg2+ (and Ca2+) site(s) in the unphosphorylated PMCa(2+)-ATPase accelerates the E2-->E1 transition and inhibits the shifting E1P--> E2P. Since with less apparent affinity La3+ slowed but did not impede phosphorylation, it seems that the sharp slowing of the rate of transformation of E1P into E2P by displacement of Mg2+ was the cause of the high-affinity inhibition of the PMCa(2+)-ATPase by La3+.