H⁺,K⁺-ATP酶催化循环中钙对镁的取代。二价阳离子两种作用的证据。

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations.

作者信息

Mendlein J, Sachs G

机构信息

Department of Physiology, UCLA School of Medicine.

出版信息

J Biol Chem. 1989 Nov 5;264(31):18512-9.

PMID:2553712

Abstract

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

摘要

为了确定二价阳离子在H⁺,K⁺-ATP酶反应机制中的作用，我们用钙替代了镁，H⁺,K⁺-ATP酶从ATP和PO₄进行磷酸化作用时需要镁。选择钙而不是其他检测过的二价阳离子（钡和锰），是因为在没有镁的情况下，钙能激活ATP水解，从[γ-³²P]ATP产生足够高的磷酸酶水平（573±51 pmol·mg⁻¹）用于研究去磷酸化作用，并且抑制K⁺刺激的ATP水解。H⁺,K⁺-ATP酶的Ca²⁺-ATP酶活性是基础Mg²⁺-ATP酶活性的40%。然而，Ca²⁺,K⁺-ATP酶活性（减去Ca²⁺基础活性）仅为Mg²⁺,K⁺-ATP酶活性的0.7%，这表明钙在激活ATP水解方面可以部分替代Mg²⁺，但在K⁺刺激ATP水解方面不行。约0.1 mM的钙抑制50%的Mg²⁺-ATP酶或Mg²⁺,K⁺-ATP酶活性。对Mg²⁺,K⁺-ATP酶活性的抑制对K⁺而言不是竞争性的。钙对Mg²⁺,K⁺活性对硝基苯磷酸酶活性的抑制对Mg²⁺而言是竞争性的，表观解离常数Ki为0.27 mM。在Ca²⁺和K⁺存在的情况下，通过吖啶橙摄取测量未检测到质子转运。在Mg²⁺和K⁺存在的情况下，Ca²⁺抑制质子转运，其表观亲和力与对Mg²⁺,K⁺-ATP酶活性的抑制相似。钙抑制的位点在囊泡外部。这些结果表明，钙通过结合在胞质二价阳离子位点来激活基础周转并抑制H⁺,K⁺-ATP酶的K⁺刺激。从5 μM [γ-³²P]ATP形成磷酸酶的准一级速率常数在有钙（0.015 s⁻¹）存在时比有镁（大于0.310 s⁻¹）时至少慢22倍。在8 mM反式-1,2-二氨基环己烷-N,N,N',N'-四乙酸（CDTA）或250 μM ATP存在的情况下，Ca.EP（在Ca²⁺存在下形成的磷酸酶）去磷酸化的速度比Mg.EP（在Mg²⁺存在下形成的磷酸酶）慢四到五倍。与超过85%的Mg.EP相比，形成的Ca.EP中约10%对100 mM KCl追踪敏感。通过比较在镁（Mg.EP）和钙（Ca.EP）存在下形成的磷酸酶的瞬态动力学，我们发现了二价阳离子在去磷酸化作用上的两种作用。（摘要截选至400字）