神经元型与非神经元型（钠钾）-ATP酶在构象平衡方面的差异。

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium.

作者信息

Matsuda T, Iwata H

机构信息

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Japan.

出版信息

Arch Biochem Biophys. 1988 Jun;263(2):323-33. doi: 10.1016/0003-9861(88)90643-1.

DOI:10.1016/0003-9861(88)90643-1

PMID:2837150

Abstract

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.

摘要

进行了多项实验以研究（Na⁺ + K⁺）-ATP 酶的两种同工酶（α⁺和α）在构象平衡方面的差异。大鼠脑（Na⁺ + K⁺）-ATP 酶比肾酶对热更不稳定。两种酶不仅受到 Na⁺和 K⁺的保护而免受热失活，而且在不同程度上也受到胆碱的保护，不过两种酶在配体保护方面存在差异。脑酶受到 Na⁺和 K⁺的部分保护而免受过 N-乙基马来酰亚胺（NEM）失活的影响，但配体对肾酶 NEM 失活的影响更为复杂。尽管配体对两种酶的热稳定性和 NEM 敏感性有不同影响，但这些影响与构象状态并非简单相关。然后研究了在 ATP、Na⁺和 Mg²⁺存在下形成的磷酸酶（EP）对 ADP 或 K⁺以及 K⁺-对硝基苯磷酸酶（pNPPase）的敏感性，以此作为两种同工酶构象平衡差异的探针。脑酶的 EP 对 ADP 部分敏感，而心脏和肾酶的 EP 则不敏感。在生理 Na⁺浓度下，脑酶和肾酶形成 E1P 的百分比分别约为总 EP 的 40 - 50%和 10 - 20%。在反应催化位点的选择性激活剂 Li⁺存在下，pNPP 的水解活性在肾酶中比在脑酶中高得多。ATP 和 Na⁺对 K⁺刺激的 pNPPase 的抑制在脑酶中比在肾酶中更大。这些结果表明，神经元和非神经元（Na⁺ + K⁺）-ATP 酶在构象平衡方面存在差异：在周转过程中，E1 或 E1P 在α⁺中可能比在α中更稳定，相反 E2 或 E2P 在后者中可能比在前者中更稳定。