Either the carboxyl- or the amino-terminal region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8).
Either the carboxyl- or the amino-terminal region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8).
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人外源 ATP 酶 (E-NTPDase 2) 的羧基或氨基末端区域赋予鸡外源 ATP-二磷酸水解酶 (E-NTPDase 8) 洗涤剂和温度敏感性。
DOI：
10.1021/bi050019k
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
A. Knowles
中科院分区：
文献类型：
--
作者：
T. Mukasa;Yonghee Lee;A. Knowles
人外源 ATP 酶 (E-NTPDase 2) 和鸡外源 ATP-二磷酸水解酶 (E-NTPDase 8) 是具有两个跨膜结构域的细胞表面核苷酸酶，N 端和 C 端各一个。 Hydrolysis of substrates occurs in active sites residing in their extracellular domains. NP-40 在温度高于 37°C 时会降低人外源 ATP 酶活性。通过凝集素和化学交联剂预先交联外源 ATP 酶，可以消除活性的降低 [Knowles, A. F. 和Chiang, W.-C. （2003）拱门。生物化学。生物物理学。 418、217-227]。相比之下，鸡胞外-ATP-二磷酸水解酶不受 NP-40 抑制，并且在 55°C 时活性大约高出 2 倍。为了确定两种 E-NTPDase 的跨膜结构域是否介导它们各自对去污剂和高温的反应，我们首先构建了一个嵌合体 (ck-hu ACR5)，其中鸡胞外-ATP-二磷酸水解酶的 C 末端被相应的人类胞外 ATP 酶区域。虽然该嵌合体表现出许多与亲本鸡胞外 ATP-二磷酸水解酶相似的酶学特征，但其对 NP-40、高温和底物的抑制与人胞外 ATP 酶相似，后者提供 C 末端，包括 C 末端跨膜结构域。此外，ConA、二琥珀酰亚胺基辛二酸酯和戊二醛对亲本酶和嵌合体的影响的比较表明，两种E-NTPDase的胞外域中发生的催化对构象限制的反应不同。第二个嵌合体（ck-hu ACR1）的酶活性也被NP-40抑制，其中鸡胞外ATP二磷酸水解酶的N端被人胞外ATP酶的相应区域取代，并且在55℃时活性较低； however, its temperature dependence differs from that of ck-hu ACR5.这些结果表明：(1) 包含两个跨膜结构域的两个 E-NTPDase 的 C 和 N 末端是决定人外源 ATP 酶对 NP-40 和高温敏感性的重要元件； (2) 单独将人外源 ATP 酶的 C 端或 N 端掺入鸡外源 ATP 二磷酸水解酶中，足以对因膜扰动而导致的 ATP 水解产生负调节； (3)两组异源跨膜结构域的相互作用并不等同，这很可能与它们的氨基酸序列不同有关。
Human ecto-ATPase (E-NTPDase 2) and chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8) are cell surface nucleotidases with two transmembranous domains, one each at the N- and C-termini. Hydrolysis of substrates occurs in active sites residing in their extracellular domains. Human ecto-ATPase activity is decreased by NP-40 and at temperatures higher than 37 degrees C. Reduction of activity is abolished by prior cross-linking of the ecto-ATPase by lectin and chemical cross-linking agents [Knowles, A. F., and Chiang, W.-C. (2003) Arch. Biochem. Biophys. 418, 217-227]. In contrast, the chicken ecto-ATP-diphosphohydrolase is not inhibited by NP-40, and activity is approximately 2-fold higher at 55 degrees C. To determine if the transmembranous domains of the two E-NTPDases mediate their respective responses to detergents and high temperature, we first constructed a chimera (ck-hu ACR5) in which the C-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase. While this chimera displays many similar enzymatic characteristics as the parental chicken ecto-ATP-diphosphohydrolase, its inhibition by NP-40, high temperature, and substrate resemble that of the human ecto-ATPase, which donates the C-terminus including the C-terminal transmembranous domain. Additionally, comparison of the effects of ConA, disuccinimidyl suberate, and glutaraldehyde on the parental enzymes and the chimera indicated that catalysis which occurs in the extracellular domains of the two E-NTPDases responds differently to conformational constraints. Enzyme activity of a second chimera (ck-hu ACR1) in which the N-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase is also inhibited by NP-40 and is less active at 55 degrees C; however, its temperature dependence differs from that of ck-hu ACR5. These results indicate that (1) the C- and N-termini of the two E-NTPDases encompassing the two transmembranous domains are important elements in determining the sensitivity of the human ecto-ATPase to NP-40 and high temperatures; (2) incorporation of either the C- or N-terminus of the human ecto-ATPase alone in the chicken ecto-ATP-diphosphohydrolase is sufficient to impart negative regulation on ATP hydrolysis due to membrane perturbation; and (3) interactions of the two sets of heterologous transmembranous domains are not equivalent, which are most likely related to their different amino acid sequences.