Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose.
The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.
Duschak
Vilma G
VG
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Av. Gral Paz y Albarellos, Parque Tecnológico Miguelete, INTI-Edificio 24, (1650) San Martín, Prov. Buenos Aires, Argentina. vduschak@iib.unsam.edu.ar
Barboza
Mariana
M
Couto
Alicia S
AS
D000327:Adsorption; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A / Q000378:metabolism*; D015891:Cystatins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010455:Peptides / Q000378:metabolism; D015800:Protozoan Proteins; D012685:Sepharose / Q000378:metabolism*; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010455:Peptides; D015800:Protozoan Proteins; D003208:Concanavalin A; D012685:Sepharose; D003546:Cysteine Endopeptidases; C063214:cruzipain