Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes
Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes
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DOI：
10.1046/j.1432-1033.2003.03542.x
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发表时间：
2003-04-01
期刊：
影响因子：
--
通讯作者：
Zimmermann, H
中科院分区：
文献类型：
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作者：
Failer, BU;Aschrafi, A;Zimmermann, H
NTPDase1和NTPDase2是两种相关的质膜定位酶，参与核苷5‘-三二磷酸的胞外降解。它们对ATP和ADP的水解率不同。这两种酶都有一个预测的跨膜结构域，分别靠近N-末端和C-末端，由携带活性部位的广泛的胞外结构域连接。我们在非洲爪哇卵母细胞中表达了鼠源性酶，并分析了它们的四级结构。应用蓝色天然PAGE和戊二醛交联剂的比较表明，天然NTPDase1和NTPDase2以低聚形式存在。通过表面碘标记证实了位于细胞表面的酶的低聚物的形成。这两种酶在低聚物结构和低聚物复合体稳定性方面存在差异。在考马斯亮蓝G-250和二硫苏糖醇存在下，NTPDase1优先以二聚体形式存在，可以解离成单体形式，而NTPDase2表现出更高的寡聚体形式，直到四聚体，对二硫苏糖醇具有很大的抗性。我们的结果进一步表明，这些酶以不同的寡聚状态存在。与NTPDase1相反，NTPDase2的底物特异性随着表达时间的延长而改变，导致ATPase/ADPase活性比从10：1下降到2.5：1，并伴随着向更高低聚状态的转变。我们的结果表明，尽管序列相同，NTPDase1和NTPDase2在寡聚结构上是不同的。寡聚体状态的动态变化可能会导致底物偏好的变化，从而影响细胞外核苷酸的原位降解模式。
NTPDase1 and NTPDase2 are two related plasma membrane-located enzymes involved in the extracellular degradation of nucleoside 5'-tri- and -diphosphates. They differ regarding their hydrolysis ratios for ATP and ADP. Both enzymes have a predicted transmembrane domain close to the N- and C-terminus, respectively, connected by an extensive extracellular domain that carries the active site. We expressed the rat-derived enzymes in Xenopus laevis oocytes and analyzed their quarternary structure. As revealed by application of blue native PAGE and a comparison of glutaraldehyde cross-linking, native NTPDase1 and NTPDase2 occur in oligomeric form. Oligomer formation of the cell surface-located pool of the enzymes was verified by surface iodination. The two enzymes differed in oligomeric structure and in oligomer complex stability. NTPDase1 preferentially occurred as a dimer that could be dissociated into monomeric forms in the presence of Coomassie Brilliant blue G-250 and dithiothreitol whereas NTPDase2 revealed higher oligomeric forms up to tetramers, largely resistant to dithiothreitol. Our results further suggest that the enzymes exist in varying oligomeric states. In contrast to NTPDase1, substrate specificity of NTPDase2 was altered with prolonged expression time, resulting in a decrease in the ATPase/ADPase activity ratio from 10 : 1 to 2.5 : 1. This was accompanied by a transition into a higher oligomeric state. Our results suggest that despite close sequence identity, NTPDase1 and NTPDase2 differ in oligomeric structure. Dynamic alterations in oligomeric state may induce changes in substrate preference and thus influence the pattern of extracellular nucleotide degradation in situ.