Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites
Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites
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DOI：
10.1007/s00436-002-0739-8
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发表时间：
2003-03-01
影响因子：
2
通讯作者：
Asai, T
中科院分区：
文献类型：
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作者：
Maeda, T;Saito, T;Asai, T
我们克隆了弓形虫丙酮酸激酶的cDNA，并获得了全长重组酶，计算分子量为57.5 kDa。预测的T.弓形虫丙酮酸激酶与柔嫩艾美耳球虫丙酮酸激酶的同源性最高（63%），与其它生物丙酮酸激酶的同源性较低（低于25%）。Southern杂交分析表明，丙酮酸激酶基因在T.弓形虫速殖子活性重组酶含有四个亚基，并产生强烈的S形饱和曲线与磷酸烯醇丙酮酸作为可变底物。果糖1，6-二磷酸是大多数物种中丙酮酸激酶的通用激活因子，但不影响该酶的活性。然而，葡萄糖6-磷酸从根本上激活酶。果糖2，6-二磷酸抑制反应速度在较高浓度的磷酸烯醇丙酮酸。这些特性表明，T.弓形虫由不寻常的磷酸化糖调节。
We have cloned a cDNA encoding Toxoplasma gondii pyruvate kinase and obtained the full-length recombinant enzyme with a calculated molecular mass of 57.5 kDa. The predicted amino acid sequence of T. gondii pyruvate kinase exhibited a highest identity (63%) to that of Eimeria tenella pyruvate kinase and a lower identity of less than 25% to the pyruvate kinases from other organisms. Southern blot analysis indicated that the pyruvate kinase gene existed as a single copy in the T. gondii tachyzoite. The active recombinant enzyme contained four subunits and produced a strongly sigmoid saturation curve with phosphoenolpyruvate as the variable substrate. Fructose 1,6-diphosphate, a general activating factor of pyruvate kinase in most species, did not affect the enzyme activity. However, glucose 6-phosphate radically activated the enzyme. Fructose 2,6-diphosphate suppressed the reaction velocity at a higher concentration of phosphoenolpyruvate. These properties indicate that pyruvate kinase activity in T. gondii is regulated by unusual phosphorylated sugars.