Trypanosoma rangeli:: Characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity
Trypanosoma rangeli:: Characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity
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DOI：
10.1016/j.exppara.2005.09.005
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发表时间：
2006-02-01
影响因子：
2.1
通讯作者：
Meyer-Fernandes, JR
中科院分区：
文献类型：
--
作者：
Fonseca, FV;de Souza, ALF;Meyer-Fernandes, JR
在这项工作中，我们描述了活的兰氏锥虫水解细胞外三磷酸腺苷（ATP）的能力。在这些完整的寄生虫中，我们在反应前后通过运动性以及台盼蓝拒染法评估了其生存能力，在没有任何二价金属离子的情况下，ATP水解水平较低（每10⁷个细胞每小时1.53 ± 0.12纳摩尔无机磷）。氯化镁（MgCl₂）可刺激ATP水解，依赖镁的外ATP酶活性为每10⁷个细胞每小时5.24 ± 0.64纳摩尔无机磷。依赖镁的外ATP酶活性与细胞密度以及时间至少在60分钟内呈线性关系。当用氯化锰（MnCl₂）替代氯化镁时，也观察到对ATP水解的这种刺激作用，但氯化钙（CaCl₂）、氯化锶（SrCl₂）和氯化锌（ZnCl₂）则无此作用。Mg - ATP²⁻的表观Km值为0.53 ± 0.11毫摩尔。兰氏锥虫依赖镁的外ATP酶活性的最适pH值在碱性范围内。这种外ATP酶活性对其他ATP酶和磷酸酶活性的抑制剂不敏感，例如寡霉素、叠氮化钠、巴弗洛霉素A1、哇巴因、呋塞米、钒酸盐、钼酸盐、氟化钠、酒石酸盐和左旋咪唑。为了证实这种依赖镁的ATP酶是一种外ATP酶，我们使用了一种不可渗透的抑制剂，即4,4'-二异硫氰基-2,2'-二磺酸（DIDS）以及苏拉明，一种P₂嘌呤受体拮抗剂和某些外ATP酶的抑制剂。这两种试剂以剂量依赖的方式抑制依赖镁²⁺的ATP酶活性。这种外ATP酶活性受到参与长红猎蝽唾液腺附着/入侵的碳水化合物以及血淋巴中循环的一种昆虫脂蛋白——载脂蛋白的刺激。（c）2005年爱思唯尔公司。保留所有权利。
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53 +/- 0.12 nmol Pi/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg-dependent ecto-ATPase activity was 5.24 +/- 0.64 nmol Pi/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2, SrCl2, and ZnCl2. The apparent K-m for Mg-ATP2- was 0.53 +/- 0.11 mM. The optimum pH for the T rangeli Mg-dependent ectoATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin Al, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levarnizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph. (c) 2005 Elsevier Inc. All rights reserved.