Cruzipain: Membrane isoforms and their expression in different stages of Trypanosoma cruzi

FABIOLA PARUSSINI; VILMA G. DUSCHAK; JUAN JOSÉ CAZZULO

Pucón

Congreso; VIII PABMB Congress in conjunction with XXXII Annual Meeting of the Argentinean Society for Biochemistry and Molecular Biology Research. XIX Annual Meeting of tha Society of Biochemistry and Moleculaar Biology of Chile; 1996

Pan-American Association for Biochemistry and Molecular Biology

Cruzipain is the major cysteine proteinase (CP) present in Trpanosoma cruzi. Its expression is developmentally regulated, levels in epimastigotes being one to two orders of magnitude higher as compared with the other parasite forms. Natural cruzipain is produced as a mixture of isoforms, probably due to the simultaneous expression of several genes. We have recently found evidence confirming the presence of a membrane isoform, which partitions in the detergent phase upon extraction with Triton X-114 and phase separation, and is liberated by treatment with phospholipase C from Bacillus cereus, thus suggesting that it is glycophosphatidyl inositol (GPI) anchored to the membrane. When the epimastigotes were disrupted by grinding with silicon carbide and the homogenate was fractionated by differential centrifugation, the putative membrane form was enriched in the microsomal fraction, which is known to consist mostly of plasma membrane fragments. Triton X-114 extracts of epimastigote, amastigote and trypomastigote forms. of the RA strain of the parasite presented this amphiphilic isoform, at different levels and with different electrophoretic mobility. Purification of total cruzipain isoforms from the three parasite stages was attained by affinity chromatography on.cystatin­Sepharose. Electrophoretic analysis of the purified  material obtained confirmed the differential expression of cruzipain isoforms along the parasite´s life cycle. Supported by SAREC (Sweden) and TDRlWHO.