镧对质膜Ca(2+)-ATP酶抑制机制的稳态前动力学研究

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

作者信息

Herscher C J, Rega A F

机构信息

Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.

出版信息

Biochemistry. 1996 Nov 26;35(47):14917-22. doi: 10.1021/bi961879r.

DOI:10.1021/bi961879r

PMID:8942656

Abstract

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+.

摘要

已知镧系元素是质膜钙泵（PMCa(2 +)-ATPase）的有效抑制剂。利用快速化学淬灭技术，在37℃下研究了LaCl₃对猪红细胞破碎完整膜上钙依赖性ATP酶（PMCa(2 +)-ATPase）水解ATP过程中发生的部分反应的影响。在磷酸化之前添加LaCl₃（K₀.₅ = 2.8 ± 0.2 μM）使E2→E1转变的kapp从14 ± 2 s⁻¹提高到23 ± 4 s⁻¹。该效应与Ca²⁺和Mg²⁺无关，就好像La³⁺以更高效率替代Mg²⁺和/或Ca²⁺来加速E1的形成。在非限制条件下，LaCl₃使静息状态下含Ca²⁺和Mg²⁺的酶中E1的表观浓度增加一倍。磷酸化过程中添加LaCl₃（K₀.₅接近20 μM）使反应的vo从300 ± 20降至60 ± 7 pmol/mg蛋白质/秒，这一速率与无Mg²⁺时相近。Mg²⁺可逆转此效应（Ca²⁺则不能），并且作为磷酸化反应激活剂的Mg²⁺的K₀.₅随LaCl₃浓度线性增加，表明La³⁺通过将Mg²⁺从激活位点取代而减缓磷酸化。如果在磷酸化之前添加，LaCl₃将EP分解的kapp降低至0.8 ± 0.1 s⁻¹，该值是无Mg²⁺时磷酸酶的特征值。此效应的K₀.₅为0.9 ± 0.5 μM LaCl₃，且随Mg²⁺浓度线性增加。如果在磷酸化之后添加，LaCl₃不会改变EP分解的90 ± 7 s⁻¹的kapp，这表明La³⁺从占据该位点会加速E1P向E2P转变的位点取代了Mg²⁺。在含有0.5 mM MgCl₂的介质中，2 μM LaCl₃迅速将PMCa(2 +)-ATPase的ATP稳态水解速率降低至接近在含LaCl₃介质中产生的EP分解速率的值。将MgCl₂增加至10 mM可在孵育的前10分钟内保护PMCa(2 +)-ATPase免受抑制。结果表明，未磷酸化的PMCa(2 +)-ATPase中La³⁺与Mg²⁺（和Ca²⁺）位点结合会加速E2→E1转变并抑制E1P→E2P转变。由于La³⁺以较低的表观亲和力减缓但未阻碍磷酸化，似乎通过取代Mg²⁺使E1P转化为E2P的速率急剧减慢是La³⁺对PMCa(2 +)-ATPase产生高亲和力抑制的原因。