Pre-Steady-State Kinetic Study of the Mechanism of Inhibition of the Plasma Membrane Ca2+-ATPase by Lanthanum

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Abstract
Lanthanides are known to be effective inhibitors of the PMCa2+-ATPase. The effects of LaCl3 on the partial reactions that take place during ATP hydrolysis by the calcium-dependent ATPase from plasma membrane (PMCa2+-ATPase) were studied at 37 °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 μM) 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 μM) 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 μM 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 μM LaCl3 lowered rapidly the rate of steady-state hydrolysis of ATP by the PMCa2+-ATPase to a value close to the rate of decomposition of EP made in medium with LaCl3. Increasing MgCl2 to 10 mM protected the PMCa2+-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 PMCa2+-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 PMCa2+-ATPase by La3+.