title: A metalloproteinase extracellularly released by Crithidia deanei.
abstract: Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.
authors.affiliation: Dept. Microbiologica Geral, Inst. Microbiologia Prof. Paulo de Góes, CCS, Cidade Universitária, Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: Claudia Masini
authors.initials: CM
authors.lastname: d'Avila-Levy
authors.forename: Rodrigo F
authors.initials: RF
authors.lastname: Souza
authors.forename: Rosana C
authors.initials: RC
authors.lastname: Gomes
authors.forename: Alane B
authors.initials: AB
authors.lastname: Vermelho
authors.forename: Marta H
authors.initials: MH
authors.lastname: Branquinha
mesh_terms: D000818:Animals; D001419:Bacteria / Q000254:growth & development; D003421:Crithidia / Q000201:enzymology* / Q000254:growth & development / Q000382:microbiology; D003470:Culture Media; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013559:Symbiosis
chemical_list: D003470:Culture Media; D011480:Protease Inhibitors; D008666:Metalloendopeptidases