Molecular organization and physiological functions of the respiratory chain specific for mitochondria from Plasmodium

1.疟原虫线粒体呼吸链的研究在显微镜下观察到疟疾寄生虫疟原虫线粒体和原生质体紧密相连。在这项研究中，我们测试了两种不同的分离技术-Percoll密度梯度离心法和荧光激活细胞器分选法在提高恶性疟原虫粗细胞器提取线粒体纯度方面的适用性。令我们惊讶的是，在每种方法中，质外体与原质质线粒体都是不可分割的。这意味着这两个原生质细胞器是相互结合的。这是疟原虫两个细胞器之间物理结合的第一个实验证据。基于氨基酸序列相似性和利用Quin…催化氧四电子还原为水的能力的寄生原生动物抗氰化交替氧化酶及其特异性抑制剂呋喃酮的研究更多的醇底物，线粒体交替氧化酶(AOX)和叶绿体末端氧化酶(PTOX)似乎是膜结合的二铁羧酸基蛋白质中的两个密切相关的成员。在目前的研究中，我们利用间日锥虫AOX(TvAOX)的高活性来研究AOX和PTOX预测的第三螺旋区域周围保守的Glu和Tyr残基的重要性。我们首先比较了TvAOX与不同类群的AOX和PTOX的氨基酸序列，然后对TvAOX的Y199和Y247氨基酸进行了丙氨酸扫描突变。我们发现，在E214A突变体中，TvAOX的泛喹酚氧化酶活性完全丧失，而突变体E215A和E216A保留了30%以上的野生型活性。在Tyr突变体中，Y221a突变体的活性也完全丧失，而Y199A、Y212A和Y247A突变体的活性相当于野生型。最后发现G1u214和Tyr221残基在AOX和PTOX之间是严格保守的。根据这些发现，AOXs和PTOXs似乎是需要保守基序E(X)EY来获得酶活性的二铁羧酸盐蛋白的一个新的亚类。较少

1. Studies on the respiratory chain specific for mitochondria from PlasmodiumThe mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques-Percoll density gradient centrifugation and fluorescence-activated organelle sorting for improving the purity of mitochondria isolated from the crude organelle preparation of P falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.2. Studies on cyanide-resistant alternative oxidases from parasitic protozoa and its spescific inhibitor, ascofuranoneBased on amino acid sequence similarity and the ability to catalyze the four-electron reduction of oxygen to water using a quin … More ol substrate, mitochondrial alternative oxidase (AOX) and plastid terminal oxidase (PTOX) appear to be two closely related members of the membrane-bound diiron carboxylate group of proteins. In the current studies, we took advantage of the high activity of Trypanosoma vivax AOX (TvAOX) to examine the importance of the conserved Glu and the Tyr residues around the predicted third helix region of AOXs and PTOXs. We first compared the amino acid sequences of TvAOX with AOXs and PTOXs from various taxa and then performed alanine-scanning mutagenesis of TvAOX between amino acids Y199 and Y247. We found that the ubiquinol oxidase activity of TvAOX is completely lost in the E214A mutant, whereas mutants E215A and E216A retained more than 30% of the wild-type activity. Among the Tyr mutants, a complete loss of activity was also observed for the Y221A mutant, whereas the activities were equivalent to wild-type for the Y199A, Y212A, and Y247A mutants. Finally, residues G1u214 and Tyr221 were found to be strictly conserved among AOXs and PTOXs. Based on these findings, it appears that AOXs and PTOXs are a novel subclass of diiron carboxylate proteins that require the conserved motif E(X)eY for enzyme activity. Less