Figure 3: In cell metabolism of pepstatin esters.
From: Esterase mutation is a mechanism of resistance to antimalarial compounds
(a) Detection of pepstatin (P) or pepstatin esters (E) by MALDI-TOF. Fractions of 3D7 lysates after incubation with pepstatin (P) or analogues were spotted onto a MALDI plate and scored for the presence or absence of compounds with appropriate mass. This MS assay is not quantitative. (b–d) MALDI-TOF spectra of HPLC fractions (C18) from whole cells extracts of 3D7 (b) or S179T mutant parasite (c,d) infected RBCs, incubated with PBE. The dominant peak of 707.0 (b) corresponds to the Na-adduct of pepstatin. The dominant peak of 766.1 (c) corresponds to the Na adduct of PBE. Pepstatin was never detected in any fractions prepared from S179T mutant parasite extracts (d). (e) Activity of recombinant PfPARE on pepstatin esters and MDTIP compounds16. Wild-type or mutant proteins were incubated with compounds and amounts of de-esterified products were determined by LC/MS (triplicate reactions). Results for incubations with wild-type enzyme are in black, with S179G mutant enzyme is in red. *Pepstatin from incubations with PM could be detected by MALDI-TOF, but not significantly by LC/MS. Error bars are s.d.
