Zeuthen Thomas, Wu Binghua, Pavlovic-Djuranovic Slavica, Holm Lars M, Uzcategui Nestor L, Duszenko Michael, Kun Jürgen F J, Schultz Joachim E, Beitz Eric
Nordic Centre for Water Imbalance Related Disorders, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Mol Microbiol. 2006 Sep;61(6):1598-608. doi: 10.1111/j.1365-2958.2006.05325.x. Epub 2006 Aug 3.
Plasmodium falciparum uses amino acids from haemoglobin degradation mainly for protein biosynthesis. Glutamine, however, is mostly oxidized to 2-oxoglutarate to restore NAD(P)H + H+. In this process two molecules of ammonia are released. We determined an ammonia production of 9 mmol h(-1) per litre of infected red blood cells in the early trophozoite stage. External application of ammonia yielded a cytotoxic IC50 concentration of 2.8 mM. As plasmodia cannot metabolize ammonia it must be exported. Yet, no biochemical or genomic evidences exist that plasmodia possess classical ammonium transporters. We expressed the P. falciparum aquaglyceroporin (PfAQP) in Xenopus laevis oocytes and examined whether it may serve as an exit pathway for ammonia. We show that injected oocytes: (i) acidify the medium due to ammonia uptake, (ii) take up [14C]methylamine and [14C]formamide, (iii) swell in solution with formamide and acetamide and (iv) display an ammonia-induced NH4+-dependent clamp current. Further, a yeast strain lacking the endogenous aquaglyceroporin (Fps1) is rescued by expression of PfAQP which provides for the efflux of toxic methylamine. Ammonia permeability was similarly established for the aquaglyceroporins from Toxoplasma gondii and Trypanosoma brucei. Apparently, these aquaglyceroporins are important for the release of ammonia derived from amino acid breakdown.
恶性疟原虫主要利用血红蛋白降解产生的氨基酸进行蛋白质生物合成。然而，谷氨酰胺大多被氧化为2-氧代戊二酸以恢复NAD(P)H + H+。在此过程中会释放出两分子氨。我们测定了早期滋养体阶段每升感染红细胞的氨产量为9 mmol h(-1)。外部施加氨产生的细胞毒性IC50浓度为2.8 mM。由于疟原虫无法代谢氨，所以必须将其输出。然而，尚无生化或基因组证据表明疟原虫拥有经典的铵转运蛋白。我们在非洲爪蟾卵母细胞中表达了恶性疟原虫水甘油通道蛋白（PfAQP），并研究它是否可作为氨的输出途径。我们发现注射后的卵母细胞：（i）由于摄取氨而使培养基酸化，（ii）摄取[14C]甲胺和[14C]甲酰胺，（iii）在含有甲酰胺和乙酰胺的溶液中肿胀，以及（iv）显示出氨诱导的NH4+依赖性钳制电流。此外，缺乏内源性水甘油通道蛋白（Fps1）的酵母菌株通过PfAQP的表达得以挽救，PfAQP可使有毒甲胺流出。对于来自刚地弓形虫和布氏锥虫的水甘油通道蛋白，也同样确定了其氨通透性。显然，这些水甘油通道蛋白对于氨基酸分解产生的氨的释放很重要。
