DPAP3 activity was measured either using the DPAP fluorogenic substrates VR-ACC[32 (link)] or FR-βNA (Sigma), or with the FY01 activity-based probe. When using FY01, samples (intact parasites, parasite lysates, insect cell supernatant, or rDPAP3 purification fractions) were labelled with 1 μM FY01 for 1 h, boiled in loading buffer, run on a SDS-PAGE gel, and the fluorescence signal measured on a PharosFX (Biorad) flatbed fluorescence scanner [18 (link)]. To determine the potency and specificity of inhibitors against DPAPs and the falcipains, parasite lysates diluted in acetate buffer (50 mM sodium acetate, 5 mM MgCl2, 5 mM DTT, pH 5.5) were pretreated for 30 min with a dose response of inhibitor followed by FY01 labelling.
When using VR-ACC (10 μM) or FR-βNA (100 μM), substrate turnover was measured on a M5e Spectramax plate reader (λBex = 355 nm/λem = 460 nm or λex = 315 nm/λem = 430 nm, respectively) in 50 mM sodium acetate, 20 mM NaCl, 5 mM DTT, and 5 mM MgCl2, pH5.5. The pH dependence of rDPAP3 was determined at 10 μM VR-ACC using a 20 mM sodium acetate, 20 mM MES and 40 mM TRIS triple buffer system containing 5 mM DTT, 0.1% CHAPS, 20 mM NaCl, and 5 mM MgCl2.
When using VR-ACC (10 μM) or FR-βNA (100 μM), substrate turnover was measured on a M5e Spectramax plate reader (λBex = 355 nm/λem = 460 nm or λex = 315 nm/λem = 430 nm, respectively) in 50 mM sodium acetate, 20 mM NaCl, 5 mM DTT, and 5 mM MgCl2, pH5.5. The pH dependence of rDPAP3 was determined at 10 μM VR-ACC using a 20 mM sodium acetate, 20 mM MES and 40 mM TRIS triple buffer system containing 5 mM DTT, 0.1% CHAPS, 20 mM NaCl, and 5 mM MgCl2.
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