（钾离子 + 氢离子）-ATP酶的研究 III. 腺苷酰亚胺二磷酸的结合

Studies on (K+ + H+)-ATPase III. Binding of adenylyl imidodiphosphate.

van de Ven F J, Schrijen J J, de Pont J J, Bonting S I

Biochim Biophys Acta. 1981 Jan 22;640(2):487-99. doi: 10.1016/0005-2736(81)90473-9.

Abstract

Adenylyl imidodiphosphate (AMPPNP) binds to (K+ + H+)-ATPase from pig gastric mucosa with a dissociation constant (Kd) of 50 microM for the AMPPNP-enzyme complex. 2. Monovalent cations reduce the amount of AMPPNP bound in the following order of effectiveness Tl+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+, Li+, choline+. 3. AMPPNP binding to the enzyme has a pH optimum at pH 7.0--7.5 in the absence of added ions, which is shifted to pH 8 upon addition of MgCl2. 4. Cyclodiaminotetraacetic acid (CDTA, Tris salt) inhibits binding of AMPPNP. This inhibition is not due to chelation of Mg2+. It may be due to direct binding of CDTA to the enzyme or to removal of stabilizing cations other than Mg2+. 5. Binding curves determined in the presence of various concentrations of Mg2+ show that at low Mg2+ concentrations (less than 0.5 mM), the apparent number of binding sites is reduced, while at higher Mg2+ concentrations (greater than or equal to 0.5 mM), the binding of AMPPNP is inhibited in a competitive way. 6. From these observations it is concluded that the enzyme has two binding sites for AMPPNP and only one for Mg-AMPPNP (or two with strong anti-cooperativity), and that Mg2+ inhibits binding of Mg-AMPPNP. This finding is interpreted in terms of a model involving a dimeric form of the enzyme.

摘要

腺苷酰亚胺二磷酸（AMPPNP）与猪胃黏膜的（K⁺ + H⁺）-ATP酶结合，AMPPNP - 酶复合物的解离常数（Kd）为50微摩尔。2. 单价阳离子按以下有效性顺序降低AMPPNP的结合量：Tl⁺＞K⁺＞Rb⁺＞Cs⁺＞Na⁺、Li⁺、胆碱⁺。3. 在不添加离子的情况下，AMPPNP与酶的结合在pH 7.0 - 7.5时有最佳pH值，加入MgCl₂后该最佳pH值移至pH 8。4. 环二氨基四乙酸（CDTA，三羟甲基氨基甲烷盐）抑制AMPPNP的结合。这种抑制不是由于Mg²⁺的螯合。它可能是由于CDTA与酶的直接结合或除去了除Mg²⁺之外的稳定阳离子。5. 在各种浓度的Mg²⁺存在下测定的结合曲线表明，在低Mg²⁺浓度（小于0.5毫摩尔）时，表观结合位点数减少，而在较高Mg²⁺浓度（大于或等于0.5毫摩尔）时，AMPPNP的结合以竞争性方式受到抑制。6. 从这些观察结果得出结论，该酶有两个AMPPNP结合位点，而Mg - AMPPNP只有一个结合位点（或两个具有强反协同性的位点），并且Mg²⁺抑制Mg - AMPPNP的结合。这一发现根据涉及该酶二聚体形式的模型来解释。