Ward D G, Cavieres J D
Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom.
J Biol Chem. 1998 Jun 5;273(23):14277-84. doi: 10.1074/jbc.273.23.14277.
The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.
钠泵的Na,K-ATP酶活性对ATP呈现出明显的多位点动力学特征，这是最小酶单位αβ原聚体所固有的特性。我们认为这应源于αβ原聚体上不同的催化位点和非催化位点，因为异硫氰酸荧光素（FITC）会阻断所有α亚基上的一个高亲和力ATP位点，但修饰后的Na,K-ATP酶对核苷酸仍保留低亲和力反应（沃德，D.G.，和卡维雷斯，J.D.（1996年）《生物化学杂志》271卷，12317 - 12321页）。我们现在发现，2'（3'）-O-（2,4,6-三硝基苯基）8-叠氮基腺苷5'-二磷酸（TNP-8N3-ADP），一种ATP的高亲和力光活化类似物，在暗光条件下的测定过程中能抑制FITC修饰酶的K⁺-磷酸酶活性。在20 mM K⁺时，抑制作用的Ki为140 μM；它需要腺嘌呤环，因为2'（3'）-O-（2,4,6-三硝基苯基）（TNP）-UDP或TNP-尿苷的效力较低，而2,4,6-三硝基苯磺酸盐则无效。在紫外光照射下，TNP-8N3-ADP会使荧光素酶的K⁺-磷酸酶活性及其被[³²P]Pi磷酸化的过程失活。光失活过程受到Na⁺或Mg²⁺的刺激，并受到K⁺或过量TNP-ADP的抑制。在存在50 mM Na⁺和1 mM Mg²⁺的情况下，TNP-8N3-ADP以15 μM的K₀.₅进行光失活。此外,TNP-8N3-ADP能使FITC修饰的、可溶解的αβ原聚体光失活，甚至比膜结合的荧光素酶更有效。这些结果有力地表明，催化性和别构性ATP位点共存于Na,K-ATP酶的αβ原聚体上。
