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.
Berthonneau
J
J
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
Rodier
M H
MH
El Moudni
B
B
Jacquemin
J L
JL
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