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. 2000 Nov 6;192(9):1289–1300. doi: 10.1084/jem.192.9.1289

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JEM000200.f6ab.jpg — Differential infectivity of TCTs overexpressing cruzipain isoforms. (A) Analysis of the cysteine protease activity present in supernatants from wild-type Dm28c TCTs. Parasite suspensions (2 × 10⁷ cells/ml) were incubated at 37°C for 2 h in DMEM without FCS. The filtered supernatants were tested for peptidase activity at 37°C using 20 μM ε-NH₂-Cap-Leu-(SBz)Cys-MCA in 50 mM Na₂HPO₄, pH 6.5, 200 mM NaCl, 5 mM EDTA, and 5 mM DTT. The graph depicts enzyme activity found in trypomastigote supernatants (Supnt) activated with DTT (▪) or pretreated with 30 μM E-64 (•). (B) Protein A–agarose beads were loaded with anti–cruzipain-1 or anti–cruzipain-2 and the immunobeads were added to supernatants obtained from wild-type TCT suspensions. After 2 h of incubation at room temperature, the immune complexes associated with the beads were washed with PBS and the initial rates of hydrolysis by enzymes associated with the solid phase were monitored in the presence or absence of 30 μM E-64, as specified above. Results (initial rates) are represented by the mean of three independent experiments. (C) The alkaline pH stability of the cysteine proteases from wild-type (WT) Dm28c TCTs or TCT-cz2 was assessed by mixing 2 μl of the Triton X-100 lysates (1 mg/ml of protein) with 100 μl of 0.1 M glycine buffer, pH 12. After 5 s, the lysates were diluted in the reaction buffer and the residual E-64–sensitive peptidase activity was measured for TCT-cz2 transfectants or wild-type parasites with ε-l-NH₂-Cap-l-(SBz)C-MCA. Gray bars depict rates obtained after alkaline treatment of lysates. The values for initial rates of hydrolysis are expressed as means ± SD of three independent experiments. (D) Invasion assays performed by adding the TCT-cz1 or TCT-cz2 transfectants to monolayers of CHO-B₂R (black bars) or CHO-mock (gray bars) in Ham's F12 medium containing 1 mg/ml of BSA in the presence of 25 μM captopril for 3 h at a parasite/host cell ratio of 2:1. Assays were carried out in the presence or absence of 100 nM HOE 140 as indicated, and the number of intracellular parasites per 100 cells was calculated. Results are given as means ± SD of three independent experiments. Statistical significance (P < 0.05) is indicated by asterisks.

graphic file with name JEM000200.f6cd.jpg — Differential infectivity of TCTs overexpressing cruzipain isoforms. (A) Analysis of the cysteine protease activity present in supernatants from wild-type Dm28c TCTs. Parasite suspensions (2 × 10⁷ cells/ml) were incubated at 37°C for 2 h in DMEM without FCS. The filtered supernatants were tested for peptidase activity at 37°C using 20 μM ε-NH₂-Cap-Leu-(SBz)Cys-MCA in 50 mM Na₂HPO₄, pH 6.5, 200 mM NaCl, 5 mM EDTA, and 5 mM DTT. The graph depicts enzyme activity found in trypomastigote supernatants (Supnt) activated with DTT (▪) or pretreated with 30 μM E-64 (•). (B) Protein A–agarose beads were loaded with anti–cruzipain-1 or anti–cruzipain-2 and the immunobeads were added to supernatants obtained from wild-type TCT suspensions. After 2 h of incubation at room temperature, the immune complexes associated with the beads were washed with PBS and the initial rates of hydrolysis by enzymes associated with the solid phase were monitored in the presence or absence of 30 μM E-64, as specified above. Results (initial rates) are represented by the mean of three independent experiments. (C) The alkaline pH stability of the cysteine proteases from wild-type (WT) Dm28c TCTs or TCT-cz2 was assessed by mixing 2 μl of the Triton X-100 lysates (1 mg/ml of protein) with 100 μl of 0.1 M glycine buffer, pH 12. After 5 s, the lysates were diluted in the reaction buffer and the residual E-64–sensitive peptidase activity was measured for TCT-cz2 transfectants or wild-type parasites with ε-l-NH₂-Cap-l-(SBz)C-MCA. Gray bars depict rates obtained after alkaline treatment of lysates. The values for initial rates of hydrolysis are expressed as means ± SD of three independent experiments. (D) Invasion assays performed by adding the TCT-cz1 or TCT-cz2 transfectants to monolayers of CHO-B₂R (black bars) or CHO-mock (gray bars) in Ham's F12 medium containing 1 mg/ml of BSA in the presence of 25 μM captopril for 3 h at a parasite/host cell ratio of 2:1. Assays were carried out in the presence or absence of 100 nM HOE 140 as indicated, and the number of intracellular parasites per 100 cells was calculated. Results are given as means ± SD of three independent experiments. Statistical significance (P < 0.05) is indicated by asterisks.