Yegutkin G G, Burnstock G
Autonomic Neuroscience Institute, Royal Free and University College Medical School, University College London, Royal Free Campus, Rowland Hill Street, NW3 2PF, London, UK.
Biochim Biophys Acta. 2000 Jun 1;1466(1-2):234-44. doi: 10.1016/s0005-2736(00)00165-6.
Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.
评估了各种嘌呤能化合物对大鼠肝细胞膜中Mg(2+)依赖性[(3)H]ATP酶促水解的抑制作用。大鼠肝酶外切ATP酶具有广泛的核苷酸水解活性，对ATP表现出米氏动力学，K(m)为368±56μM，且对离子交换和细胞内ATP酶的经典抑制剂不敏感。P2拮抗剂和二腺苷四磷酸(Ap(4)A)对外切ATP酶活性具有渐进性和非竞争性抑制作用，抑制效力的顺序如下：苏拉明(pIC(50)，4.570)>活性蓝2(4.297)&z.Gt;Ap(4)A(3.268)>磷酸吡哆醛-6-偶氮苯基-2'，4'-二磺酸(PPADS)(2.930)。缓慢水解的P2激动剂ATPγS、ADPβS、α，β-亚甲基ATP和β，γ-亚甲基ATP以及二腺苷多磷酸Ap(3)A和Ap(5)A在10(-4)-10(-3)M浓度范围内对酶活性没有任何抑制作用。[(3)H]ATP代谢产物形成的薄层色谱分析表明肝表面存在其他酶活性(外切ADP酶和5'-核苷酸酶)，它们与外切ATP酶协同参与核苷酸水解，通过ATP→ADP→AMP→腺苷的逐步反应。在存在外切ATP酶抑制剂苏拉明、Ap(4)A和PPADS的情况下，仍会出现类似的[(3)H]ATP顺序去磷酸化模式，但最终反应产物腺苷的出现明显延迟。相比之下，在活性蓝2存在的情况下，[(3)H]ATP的水解仅遵循ATP→ADP模式，随后的代谢产物AMP和腺苷的形成几乎被消除。这些数据表明，尽管外切ATP酶的核苷酸结合位点与P2受体的不同，但一些嘌呤能激动剂和拮抗剂可以通过非特异性抑制随后的嘌呤分解代谢途径来增强细胞对细胞外ATP的反应。
