从狗肾中分离出的 Na+-K+ 泵磷酸化形式中钴离子的封闭。

Occlusion of cobalt ions within the phosphorylated forms of the Na+-K+ pump isolated from dog kidney.

作者信息

Richards D E

机构信息

Physiological Laboratory, Cambridge.

出版信息

J Physiol. 1988 Oct;404:497-514. doi: 10.1113/jphysiol.1988.sp017302.

DOI:10.1113/jphysiol.1988.sp017302

PMID:2855351

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1190838/

Abstract

Co2+ ions can replace Mg2+ ions as co-factors for the Na+-K+ pump purified from dog kidney outer medulla. The evidence comes from (a) measurement of ouabain-sensitive Na+,K+-ATPase activity, (b) measurement of ATP-dependent 22Na uptake catalysed by the Na+-K+ pump reconstituted into phospholipid vesicles, (c) measurements of phosphorylation of the Na+-K+ pump either in the presence of ATP and sodium ions or in the presence of inorganic phosphate, and (d) measurement of occlusion of rubidium ions through the route involving phosphorylation and dephosphorylation. 2. Purified Na+,K+-ATPase incubated in the presence of ATP, Na+ ions and [60Co]CoCl2, can carry occluded Co2+ ions through a cation-exchange resin. The enzyme fails to occlude the divalent cation (i) if ADP replaces ATP, (ii) if the enzyme is heat-inactivated, (iii) if the enzyme is inactivated by treatment with fluorescein isothiocyanate, (iv) if K+ replaces Na+ in the incubation medium, (v) if Na+ ions are omitted, and (vi) if Mg2+ ions are added in a sufficient concentration. 3. The amount of occluded Co2+ ions is unaffected by pre-treatment of the Na+,K+-ATPase with oligomycin, which stabilizes the phosphoenzyme in the E1P form. 4. The addition of K+ ions to Na+,K+-ATPase that has been phosphorylated in the presence of ATP, Na+ ions and [60Co]CoCl2 releases the occluded Co2+ ions from the enzyme. Under those conditions, K+ ions accelerate the hydrolysis of the phosphoenzyme, and become occluded in the resulting dephosphoenzyme. 5. The stoichiometry of Co2+ ion occlusion is about one occluded Co2+ ion per phosphorylation site. 6. These results support the hypothesis that, in the normal working of the Na+-K+ pump, Mg2+ ions are trapped in the phosphorylated forms of the enzyme, and are released by a K+-dependent dephosphorylation reaction.

摘要

Co²⁺离子可以取代Mg²⁺离子，作为从犬肾外髓质纯化的Na⁺-K⁺泵的辅助因子。证据来自于：(a) 哇巴因敏感的Na⁺,K⁺-ATP酶活性的测定；(b) 重组到磷脂囊泡中的Na⁺-K⁺泵催化的ATP依赖性²²Na摄取的测定；(c) 在ATP和钠离子存在下或在无机磷酸盐存在下对Na⁺-K⁺泵磷酸化的测定；以及(d) 通过涉及磷酸化和去磷酸化途径的铷离子封闭的测定。2. 在ATP、Na⁺离子和[⁶⁰Co]CoCl₂存在下孵育的纯化Na⁺,K⁺-ATP酶，可以携带封闭的Co²⁺离子通过阳离子交换树脂。如果（i）ADP取代ATP，（ii）酶被热灭活，（iii）酶用异硫氰酸荧光素处理而失活，（iv）孵育介质中K⁺取代Na⁺，（v）省略Na⁺离子，以及（vi）加入足够浓度的Mg²⁺离子，则该酶不能封闭二价阳离子。3. 用寡霉素预处理Na⁺,K⁺-ATP酶不影响封闭的Co²⁺离子的量，寡霉素可使磷酸酶稳定在E1P形式。4. 向在ATP、Na⁺离子和[⁶⁰Co]CoCl₂存在下已被磷酸化的Na⁺,K⁺-ATP酶中加入K⁺离子，会从酶中释放出封闭的Co²⁺离子。在这些条件下，K⁺离子加速磷酸酶的水解，并在产生的去磷酸酶中被封闭。5. Co²⁺离子封闭的化学计量约为每个磷酸化位点一个封闭的Co²⁺离子。6. 这些结果支持以下假设：在Na⁺-K⁺泵的正常工作中，Mg²⁺离子被困在酶的磷酸化形式中，并通过依赖于K⁺的去磷酸化反应释放。