Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family
Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family
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DOI：
10.1042/0264-6021:3510639
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发表时间：
2000-11-01
影响因子：
4.1
通讯作者：
Zimmermann, H
中科院分区：
文献类型：
--
作者：
Braun, N;Fengler, S;Zimmermann, H
我们已经分离并鉴定了编码核苷三磷酸二磷酸水解酶6（NTPDase 6）的cDNA，NTPDase 6是胞外核苷三磷酸二磷酸水解酶家族的新成员。该cDNA的开放阅读框为1365 bp，编码455个氨基酸残基的蛋白质，分子量为49971 Da。它与人CD 39 L2序列具有86%的序列同一性，与两种相关的人和鼠核苷二磷酸酶（CD 39 L4，NTPD酶5/ER-UD 4）的序列分别具有48%和51%的序列同一性。mRNA的表达在所有组织的调查，揭示了两个主要的成绩单具有不同的丰度。PCR分析表明为单一开放阅读框。在中国仓鼠卵巢（CHO）和PC 12细胞中表达Myc-His-taged NTPDase 6用于免疫分析和蛋白分离。该蛋白质包含在转染的CHO细胞的膜组分中，并以可溶形式存在于细胞培养上清液中。NTPD酶6优先水解核苷5 '-二磷酸。在不同底物条件下，酶活力大小顺序为GDP > IDP >> UDP，CDP >> ADP.核苷5 '-三磷酸仅在较小程度上水解，并且未观察到核苷5'-单磷酸的水解。该酶被Ca 2+和Mg 2+强烈且同等地激活，并且对GDP具有211 μ M的Km。转染CHO和PC 12细胞的免疫组化分析表明，NTPDase 6与高尔基体，并在很小程度上也与质膜。该酶可能支持高尔基体中的糖基化反应，并且当从细胞中释放时，可能催化细胞外核苷酸的水解。
We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% acid 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP > IDP >> UDP, CDP >> ADP. Nucleoside 5'-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly and equally activated by Ca2+ and Mg2+ and had a K-m for GDP of 211 muM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides.