Toxoplasma gondii: purification and characterization of an immunogenic metallopeptidase.
A Toxoplasma gondii aminopeptidase specific for the fluorogenic substrate L-arginine 7-amino-4-methylcoumarin was identified in cell-free extract. This enzyme was purified by high-performance liquid chromatography using first size exclusion, then anion exchange, followed by a second size exclusion. The purified enzyme exhibited a pl of 4.7 by chromatofocusing and had an apparent molecular weight of 110 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The purification factor was 80.9 and the yield was 14%. The optimal activity was at pH 7.4 and was strongly inhibited by EDTA and o-phenanthroline. Antibodies against this T. gondii metallopeptidase were detected by immunoprecipitation and immunoblotting in human sera obtained from patients undergoing toxoplasmosis.
Laboratoire de Parasitologie et Mycologie Médicales, Unité de Recherche en Biologie Parasitaire, C.H.U. La Milétrie, BP. 577, Poitiers Cedex, 86021, France. parasitologie@chu-poitiers.fr
J
J
Berthonneau
M H
MH
Rodier
B
B
El Moudni
J L
JL
Jacquemin
D000626:Aminopeptidases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D007526:Isoelectric Point; D011233:Precipitin Tests; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology*
D000913:Antibodies, Protozoan; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D000626:Aminopeptidases