Sangolgi Prakash B, Balaji Chinthapalli, Dutta Sneha, Jindal Nitin, Jarori Gotam K
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
Protein Expr Purif. 2016 Jan;117:17-25. doi: 10.1016/j.pep.2015.08.028. Epub 2015 Sep 1.
Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. ∼ 30-40 mg pure recombinant enzyme with a specific activity of 12.6 units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH ∼ 9. Kinetic measurements gave KmNAD=0.28 ± 0.3 mM and KmG3P=0.25 ± 0.03 mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW ∼ 27, ∼ 37 and ∼ 51 kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the ∼ 51 kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membranes) examined had Gapdh associated with them. Each fraction had multiple molecular species associated with them. Such species could arise only by multiple post-translational modifications. Structural heterogeneity observed among molecular species of PyGapdh and their diverse subcellular distribution, supports the view that Gapdh is likely to have multiple non-glycolytic functions in the parasite and could be an effective target for anti-malarial chemotherapeutics.
疟原虫属在人类红细胞内进行无性繁殖期间，完全依靠糖酵解来满足其能量需求，这使得该途径的酶成为潜在的药物靶点。我们已克隆、过量表达并纯化了恶性疟原虫甘油醛-3-磷酸脱氢酶（PfGapdh），用于其动力学和结构表征。从一升大肠杆菌培养物中可获得约30 - 40毫克具有12.6单位/毫克比活性的纯重组酶。该酶是一种同四聚体，最适pH约为9。动力学测量得出KmNAD = 0.28 ± 0.3 mM，KmG3P = 0.25 ± 0.03 mM。在小鼠体内产生的多克隆抗体显示出高特异性，这从它们与兔肌肉Gapdh无反应性中可以明显看出。约氏疟原虫细胞提取物的蛋白质印迹显示在分子量约为27 kDa、37 kDa和51 kDa处有三条带。通过液相色谱-电喷雾电离质谱法确认了所有三条带中均存在PyGapdh。有趣的是，约51 kDa的形式仅存在于提取物的可溶部分中。使用差异去污剂分级分离法研究了约氏疟原虫中Gapdh的亚细胞分布。每个分级部分在二维凝胶上进行分析，并通过蛋白质印迹进行可视化。所检测的所有四个亚细胞部分（即细胞质、细胞核、细胞骨架和细胞膜）均与Gapdh相关联。每个部分都有多种与之相关的分子种类。这些种类只能通过多种翻译后修饰产生。在PyGapdh分子种类中观察到的结构异质性及其多样的亚细胞分布，支持了这样一种观点，即Gapdh在寄生虫中可能具有多种非糖酵解功能，并且可能是抗疟化学治疗的有效靶点。
