CHARACTERIZATION OF A TRYPANOSOMA-CRUZI ACIDIC 30 KDA CYSTEINE PROTEASE

A novel proteolytic activity was identified in epimastigote, amastigot e and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate -Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin Epimastig otes showed enzyme activity to be 2-fold higher than amastigotes and t rypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diet hylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concana valin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electroph oresis (PAGE), under reducing conditions. Its optimal enzymatic activi ty on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease sho wed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum al bumin (BSA) and human type I collagen. The N-terminal amino acid seque nce of the protease shows similarity to the mammalian cathepsin B prot ease. (C) 1998 Elsevier Science B.V. All rights reserved.