Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites
Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites
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DOI：
10.1042/0264-6021:3470243
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发表时间：
2000-04-01
影响因子：
4.1
通讯作者：
Docampo, R
中科院分区：
文献类型：
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作者：
Marchesini, N;Luo, SH;Docampo, R
用荧光钙指示剂fura-2 acetoxymethyl ester负载伯氏疟原虫滋养体，以测量其细胞内Ca 2+浓度（[Ca 2 +]（1））。[Ca2+]（1），在肌质/内质网Ca ~（2+）-ATP酶抑制剂毒胡萝卜素存在下增加。滋养体还具有大量的Ca 2+储存在酸性隔室中。这表现在：（1）[Ca 2 +]的增加（1）由巴弗洛霉素A、尼日利亚菌素、莫能菌素或弱碱NH 4Cl诱导，在细胞外Ca 2+名义上不存在的情况下，和（2）离子霉素的作用，其不能将Ca 2+从酸性细胞器中取出，并且在通过加入巴弗洛霉素A（1）、尼日利亚菌素、莫能菌素或NH 4Cl使该室碱化后更有效。无机PPi促进滋养体细胞匀浆中亚细胞区室的酸化。PPi驱动的质子梯度通过添加K+/H+交换剂尼日利亚菌素（nigericin）而崩溃，并通过PPi类似物氨基亚甲基二膦酸盐（AMDP）消除。PPi水解和质子转运均依赖于K+，Na+对PPi水解和质子转运有部分抑制作用。PPi水解是敏感的，以剂量依赖性的方式AMDP，亚氨基二磷酸，氟化钠，二环己基马来酰亚胺和硫醇试剂，N-乙基马来酰亚胺。免疫荧光显微镜使用的植物液泡焦磷酸酶（V-H+-PPase）的保守肽序列提出的抗体表明，质子焦磷酸酶位于细胞内液泡和滋养体的质膜。AMDP引起[Ca 2 +]增加，在细胞外Ca 2+名义上不存在的情况下，离子霉素在用AMDP处理细胞后从该酸性细胞内室释放Ca 2+方面更有效。两者合计，这些结果表明，在疟疾寄生虫的酸钙体与锥虫和刚地弓形虫中描述的那些相似的特性的存在，和共定位的V-H+-PPase和V-H+-ATP酶在这些细胞器。
Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca2+ concentration ([Ca2+](1)). [Ca2+](1), was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by: (1) the increase in [Ca2+](1) induced by bafilomycin A,, nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2+, and (2) the effect of ionomycin, which cannot take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH4CI. Inorganic PPi promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PPi collapsed by addition of the K+/H+ exchanger, nigericin, and eliminated by the PPi analogue, amino-methylenediphosphonate (AMDP). Both PPi hydrolysis and proton transport were dependent upon K+, and Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H+-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca2+], in the nominal absence of extracellular Ca2+ Ionomycin was more effective in releasing Ca2+ from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H+-PPase and V-H+-ATPase in these organelles.