| Abstrakt: |
Proteinase activities and megasomes were examined in axenically cultivated amastigotelike 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 amastigotelike 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, ophenanthrolinesensitive activities; and (b) 23 to 40kDa cysteine proteinases, of which those resolving as 35 to 40kDa bands in gelatin gels were more clearly visualized in lysates of cultured amastigotelike forms. Incubation of both kinds of amastigotes with 0.25 to 1.0 µM of either ZPheAIaCHN2 or ZTyrAIaCHN2 selectively inactivated cysteine proteinases, but not the 35 to 40kDa activities, which, again, were detected with higher intensity in cultured amastigote-like forms. The expression of the 35 to 40kDa proteinases progressively increased when promastigotes were allowed to transform into amastigotelike forms or when lesion amastigotes were incubated at 34°C for different time periods prior to exposure to ZPheAIaCHN2; activities comparable to those of amastigotelike forms were attained within 24 to 48 hr. The activities resistant to ZPheAIaCHN2 in vivo were fully inhibited by E64 or ZPheAIaCHN2 during gelatin digestion, suggesting that the 35 to 40kDa proteinases were mainly inactive before cell lysis. The presence of cycloheximide (at 10, 50, and 100 µg/ml) during the pulse with ZPheAIaCHN2 abolished the 35 to 40kDa activities of lesion amastigotes and significantly reduced gelatin digestion by the similar enzymes of cultured amastigotelike forms. In the latter, the 35 to 40kDa proteinases were no more detected when cycloheximide was given 60 min prior to ZPheAIaCHN2. The results indicate higher rates of synthesis of the 35 to 40kDa enzymes, and the existence of a more representative pool of inactive enzyme precursors, in cultured amastigotelike forms.Copyright 1993, 1999 Academic Press |
