DETECTION OF A PROTEINASE COMMON TO EPIMASTIGOTE, TRYPOMASTIGOTE AND AMASTIGOTE OF DIFFERENT STRAINS OF TRYPANOSOMA-CRUZI

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  • 1 January 1981
    • journal article
    • research article
    • Vol. 32  (2) , 87-92
Abstract
T. cruzi epimastigote lysates presented proteolytic activity both at pH 7.0 (Km = 2.5 mg casein/ml, Km = 12.2 mg Hb/ml) and at pH 3.0 (Km = 2.5 mg Hb/ml). A proteinase was isolated from these lysates by using 3 different steps: Precipitation at -20.degree. C, pH 4.5, with 80% acetone; Sephadex G200 chromatography; and affinity chromatography on columns of Sepharose 4B coupled to p-aminophenylmercuriacetate. The isolated proteinase, which probably is an SH-dependent enzyme, was able to hydrolyze Hb at pH 3.0 and both casein and Hb at pH 7.0 but was unable to hydrolyze the esterase synthetic substrate tested. A single enzyme of MW 60,000 could be detected in purified preparations when analyzed by disc gel electrophoresis, crossed immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis and Sephadex chromatography. Antibodies induced by the purified proteinase, shown to be specific for proteinase molecules by line immunoelectrophoresis experiments, reacted with epimastigota of the Y strain and with trypomastigota and amastigota of 5 different strains tested. EM observations of peroxidase-labeled preparations indicated that the proteinase could be found at the surface of different forms of the parasite.