Bermudes D, Peck K R, Afifi M A, Beckers C J, Joiner K A
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022.
J Biol Chem. 1994 Nov 18;269(46):29252-60.
The obligate intracellular parasite Toxoplasma gondii produces a nucleoside triphosphate hydrolase (NTPase) (nucleoside-triphosphatase, EC 3.6.1.15) activable by dithiol-containing compounds. We have isolated the genomic DNA for the NTPase from the RH strain of Toxoplasma and determined the nucleotide sequence of three tandemly arranged open reading frames termed NTP1, NTP2, and NTP3. We have also isolated and sequenced cDNAs for NTP1 and NTP3; no cDNA for NTP2 was obtained. The two cDNA clones encode proteins that are more than 97% identical at the amino acid level but significantly differ within two small domains, indicating the presence of NTPase isoforms. Both possess N-terminal signal sequences and two regions with partial homology to certain known ATP binding motifs: the glycine-rich loop common to many ATP binding proteins and the beta-phosphate binding domain found in the hexokinase-actin-hsp70 family. Antiserum against a NTP1-fusion protein immunoprecipitated NTPase activity from extracellular parasites that was increased in activity by treatment with dithiothreitol, confirming the identity of the cloned genes. By immunofluorescence, the NTPase is located in vesicular structures within the parasite, and in infected cells it is secreted into the vacuolar space and becomes partially associated with the parasitophorous vacuolar membrane. Since the vacuolar membrane is freely permeable to small molecules of < 1300 Da, host cell ATP may serve as a substrate for the NTPase and supply the energy for parasite-directed processes in the vacuolar space.
专性细胞内寄生虫刚地弓形虫产生一种可被含二硫醇的化合物激活的核苷三磷酸水解酶（NTPase）（核苷 - 三磷酸酶，EC 3.6.1.15）。我们从弓形虫的RH株中分离出了NTPase的基因组DNA，并确定了三个串联排列的开放阅读框（称为NTP1、NTP2和NTP3）的核苷酸序列。我们还分离并测序了NTP1和NTP3的cDNA；未获得NTP2的cDNA。这两个cDNA克隆编码的蛋白质在氨基酸水平上有超过97%的同一性，但在两个小结构域内有显著差异，表明存在NTPase同工型。两者都具有N端信号序列和与某些已知ATP结合基序部分同源的两个区域：许多ATP结合蛋白共有的富含甘氨酸的环以及己糖激酶 - 肌动蛋白 - hsp70家族中发现的β - 磷酸结合结构域。针对NTP1融合蛋白的抗血清从细胞外寄生虫中免疫沉淀出NTPase活性，用二硫苏糖醇处理后活性增加，证实了克隆基因的同一性。通过免疫荧光法，NTPase位于寄生虫内的泡状结构中，在感染细胞中它被分泌到液泡空间并部分与寄生泡膜结合。由于液泡膜对分子量小于1300 Da的小分子是自由通透的，宿主细胞ATP可能作为NTPase的底物，并为液泡空间中寄生虫定向的过程提供能量。
