title: Leishmania mexicana: proteinase activities and megasomes in axenically cultivated amastigote-like forms.
abstract: Proteinase activities and megasomes were examined in axenically cultivated amastigote-like forms, freshly isolated lesion amastigotes, and promastigotes. Megasomes were absent in promastigotes and present in both amastigote stages, but they seemed to be less numerous and more homogeneous in cultured amastigote-like forms. Contrasting with the poor detection of proteinase activities in promastigote lysates, both types of amastigotes shared multiple proteinases, which were classified in two groups: (a) 60 to > 100 kDa, o-phenanthroline-sensitive activities; and (b) 23- to 40-kDa cysteine proteinases, of which those resolving as 35- to 40-kDa bands in gelatin gels were more clearly visualized in lysates of cultured amastigote-like forms. Incubation of both kinds of amastigotes with 0.25 to 1.0 microM of either Z-Phe-AlaCHN2 or Z-Tyr-AlaCHN2 selectively inactivated cysteine proteinases, but not the 35- to 40-kDa activities, which, again, were detected with higher intensity in cultured amastigote-like forms. The expression of the 35- to 40-kDa proteinases progressively increased when promastigotes were allowed to transform into amastigote-like forms or when lesion amastigotes were incubated at 34 degrees C for different time periods prior to exposure to Z-Phe-AlaCHN2; activities comparable to those of amastigote-like forms were attained within 24 to 48 hr. The activities resistant to Z-Phe-AlaCHN2 in vivo were fully inhibited by E-64 or Z-Phe-AlaCHN2 during gelatin digestion, suggesting that the 35- to 40-kDa proteinases were mainly inactive before cell lysis. The presence of cycloheximide (at 10, 50, and 100 micrograms/ml) during the pulse with Z-Phe-AlaCHN2 abolished the 35- to 40-kDa activities of lesion amastigotes and significantly reduced gelatin digestion by the similar enzymes of cultured amastigote-like forms. In the latter, the 35- to 40-kDa proteinases were no more detected when cycloheximide was given 60 min prior to Z-Phe-AlaCHN2. The results indicate higher rates of synthesis of the 35- to 40-kDa enzymes, and the existence of a more representative pool of inactive enzyme precursors, in cultured amastigote-like forms.
authors.affiliation: Departamento de Parasitologia, Universidade de São Paulo, Brazil.
authors.forename: E M
authors.initials: EM
authors.lastname: Pral
authors.forename: A T
authors.initials: AT
authors.lastname: Bijovsky
authors.forename: J M
authors.initials: JM
authors.lastname: Balanco
authors.forename: S C
authors.initials: SC
authors.lastname: Alfieri
mesh_terms: D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D007894:Leishmania mexicana / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D015388:Organelles / Q000648:ultrastructure*; D013696:Temperature
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; C024918:benzyloxycarbonylphenylalanylalanine diazomethyl ketone; C057752:benzyloxycarbonyltyrosylalanine diazomethane; D003978:Diazomethane; D003513:Cycloheximide; D010450:Endopeptidases; D003546:Cysteine Endopeptidases