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Leishmania mexicana: molecular cloning and characterization of enolase.
- Source :
-
Experimental parasitology [Exp Parasitol] 2007 Jul; Vol. 116 (3), pp. 241-51. Date of Electronic Publication: 2007 Jan 23. - Publication Year :
- 2007
-
Abstract
- The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.
- Subjects :
- Amino Acid Sequence
Animals
Antibodies, Protozoan immunology
Base Sequence
Blotting, Western
Cell Membrane Permeability drug effects
Cloning, Molecular
Digitonin pharmacology
Electrophoresis, Polyacrylamide Gel
Fluorescent Antibody Technique
Indicators and Reagents pharmacology
Kinetics
Leishmania mexicana genetics
Leishmania mexicana immunology
Mice
Molecular Sequence Data
Phosphopyruvate Hydratase biosynthesis
Phosphopyruvate Hydratase isolation & purification
Rabbits
Recombinant Proteins biosynthesis
Recombinant Proteins chemistry
Recombinant Proteins genetics
Sequence Alignment
Leishmania mexicana enzymology
Phosphopyruvate Hydratase chemistry
Phosphopyruvate Hydratase genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0014-4894
- Volume :
- 116
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Experimental parasitology
- Publication Type :
- Academic Journal
- Accession number :
- 17382932
- Full Text :
- https://doi.org/10.1016/j.exppara.2007.01.008