Abstract
We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg2+ requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, orp-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 ± 3.0 mM) and long chain polyP (55.9 ± 5.6 mM) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.
| Original language | English |
|---|---|
| Pages (from-to) | 50899-50906 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 277 |
| Issue number | 52 |
| DOIs | |
| State | Published - Dec 27 2002 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate. / Rodrigues, Claudia D; Ruiz, Felix A.; Vieira, Mauricio; Hill, Janet E.; Docampo, Roberto.
In: Journal of Biological Chemistry, Vol. 277, No. 52, 27.12.2002, p. 50899-50906.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate
AU - Rodrigues, Claudia D
AU - Ruiz, Felix A.
AU - Vieira, Mauricio
AU - Hill, Janet E.
AU - Docampo, Roberto
PY - 2002/12/27
Y1 - 2002/12/27
N2 - We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg2+ requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, orp-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 ± 3.0 mM) and long chain polyP (55.9 ± 5.6 mM) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.
AB - We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg2+ requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, orp-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 ± 3.0 mM) and long chain polyP (55.9 ± 5.6 mM) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.
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UR - http://www.scopus.com/inward/citedby.url?scp=0037185015&partnerID=8YFLogxK
U2 - 10.1074/jbc.M208940200
DO - 10.1074/jbc.M208940200
M3 - Article
VL - 277
SP - 50899
EP - 50906
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 52
ER -