Characterisation of a Trypanosoma cruzi acidic 30 kDa cysteine protease.
A novel proteolytic activity was identified in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Epimastigotes showed enzyme activity to be 2-fold higher than amastigotes and trypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diethylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concanavalin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), under reducing conditions. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease showed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum albumin (BSA) and human type I collagen. The N-terminal amino acid sequence of the protease shows similarity to the mammalian cathepsin B protease.
Garcia
M P
MP
Departamento de Biologia Celular, Universidade de Brasília, Brasília-DF, Brazil.
Nóbrega
O T
OT
Teixeira
A R
AR
Sousa
M V
MV
Santana
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose; D003094:Collagen / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development
D015853:Cysteine Proteinase Inhibitors; D012710:Serum Albumin, Bovine; D003094:Collagen; D003546:Cysteine Endopeptidases