Fang Jianmin, Ruiz Felix A, Docampo Melissa, Luo Shuhong, Rodrigues Juliany C F, Motta Lucimar S, Rohloff Peter, Docampo Roberto
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Paul D. Coverdell Biomedical and Health Sciences Center, Athens, Georgia 30602, USA.
J Biol Chem. 2007 Nov 2;282(44):32501-10. doi: 10.1074/jbc.M704841200. Epub 2007 Sep 7.
We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.
我们报道了克氏锥虫外切多聚磷酸酶（TcPPX）的克隆、表达、纯化及特性分析。该基因产物（TcPPX）含有383个氨基酸，分子量为43.1 kDa。TcPPX与大多数外切多聚磷酸酶不同，它更倾向于作用短链多聚磷酸（多聚P）。在大肠杆菌中对TcPPX进行异源表达产生了一种功能性酶，该酶的最适pH呈中性，并且受到低浓度Zn2+、高浓度碱性氨基酸（赖氨酸和精氨酸）以及肝素的显著抑制。TcPPX是一种持续性酶，不水解ATP、焦磷酸或对硝基苯磷酸，尽管它能非常有效地水解鸟苷5'-四磷酸。TcPPX的过表达导致总短链多聚P显著减少，长链多聚P部分减少。这伴随着低渗应激后调节性容积减小的延迟。这些结果支持了多聚P在克氏锥虫渗透调节中的作用。
