On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli

119

Section: Fig 2 Expression and Subcellular Localization Of Tbvsp1mentioning

confidence: 80%

“…This exopolyphosphatase is active at pH 7.5 and is inhibited by CaCl 2 (40,41). To avoid polyP 3 accumulation in the cell compartment where polyP hydrolysis is occurring and to produce Pi, polyP 3 should be further hydrolyzed.…”

Section: Fig 2 Expression and Subcellular Localization Of Tbvsp1mentioning

confidence: 99%

A Pyrophosphatase Regulating Polyphosphate Metabolism in Acidocalcisomes Is Essential for Trypanosoma brucei Virulence in Mice

Lemercier

Espiau

Ruiz

et al. 2004

119

“…PPase and Exopolyphosphatase Activity-0.020 -2 g of desalted nLaVSP1 and mLaVSP1 proteins were incubated at 25°C for 1 min to hydrolyze PP i and 2 min for either polyP 3 or polyP 28 . The reaction was stopped, and released P i was quantified as described by Shatton et al (21).…”

Section: Methodsmentioning

confidence: 99%

“…MES/NaOH buffer was used from pH 5.5 to 6.5 and Tris-HCl from pH 7.0 to 8.8. Before use, polyP 28 was purified on G25 Sephadex.…”

Section: Methodsmentioning

confidence: 99%

A Soluble Pyrophosphatase, a Key Enzyme for Polyphosphate Metabolism in Leishmania

Espiau

Lemercier

Ambit

et al. 2006

“…Genes encoding for exopolyphosphatases from Saccharomyces cerevisiae (17), Trypanosoma brucei (18), and Leishmania major (19) have been cloned and expressed in Escherichia coli, whereas a gene encoding an endopolyphosphatase from S. cerevisiae (20) is the only one that has been cloned and expressed. The L. major exopolyphosphatase (LmPPx) has been characterized recently and demonstrated to be located in acidocalcisomes and in the cytosol (19).…”

mentioning

confidence: 99%

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

Fang

Ruiz²,

Docampo

et al. 2007

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 Zn 2؉ , 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 longchain 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. Polyphosphate (poly P)8 is a ubiquitous polymer of a few to several hundred orthophosphate residues linked by high-energy phosphoanhydride bonds. Poly P is found in the environment and in all life forms, where it is involved in a number of functions (1, 2). Poly P is essential for bacterial responses to stresses and starvation, as well as for survival and virulence (1, 2). Similar functions in adaptation to stress have been attributed to poly P in eukaryotic cells such as yeast (3, 4), fungi (5), and algae (6 -8). Poly P is also involved in blood clotting (9), eukaryotic cell proliferation (10, 11), and induction of apoptosis in plasma and myeloma cells (12).In Trypanosoma cruzi, the etiologic agent of Chagas disease, most poly P is accumulated in acidocalcisomes. Acidocalcisomes are acidic, calcium-containing organelles that have been demonstrated to be involved in osmoregulation (13-16). Rapid hydrolysis of acidocalcisome poly P occurs when epimastigotes of T. cruzi are exposed to hyposmotic stress (16) resulting in increased osmolarity and swelling of the organelle (15). In addition, a microtubule and cAMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of an aquaporin results in water movement and a regulatory volume decrease (15), indicating a link between acidocalcisomes and osmotic homeostasis.In eukaryotic cells the hydrolysis of poly P is performed by the action of endopolyphosphatases and exopolyphosphatases (1, 2). Genes encoding for exopolyphosphatases from Saccharomyces cerevisiae (17), Trypanosoma brucei (18), and Leishmania major (19) have been cloned and expressed in Escherichia coli, whereas a gene encoding an endopolyphosphatase from S. cerevisiae (20) is the only one that has been cloned and expressed. The L. major exopolyphosphatase (LmPPx) has been characterized recently and demonstrated to be located in acidocalcisomes and in the cytosol (19). An exopolyphosphatase activity has also been detected in acidocalcisomes of T. cruzi (16). The crystal structures...