Trans epoxysuccinyl l leucylamido 4 guanidino butane e64

The fibrinolytic enzyme type was determined by evaluating the effects of multifarious inhibitors on the fibrinolytic activity. Briefly, the purified enzyme solution (50 IU/ml) was pre-incubated at 37 °C for 30 min separately with each of the following inhibitors: 5.0 mM serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Sigma, Beijing, China), 5.0 mM metalloprotease inhibitor ethylene diamine tetraacetic acid (EDTA; Sigma, Beijing, China), 10 µM aspartic protease inhibitor pepstatin A (Sigma, Beijing, China), 1.0 mM cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64; Sigma, Beijing, China), and 1.0 mM aminopeptidase inhibitor bestatin (Sigma, Beijing, China). To measure the effects of metal ions on the activity, the fibrinolytic enzyme (50 IU/ml) was pre-incubated separately with different metal ions (final concentration 5 mM) at pH 7.5 and 37 °C for 30 min. The enzyme solution without added ions was used as a control.

Experiments were performed with the 3D7 laboratory strain of P. falciparum. Parasitophorous-vacuole-enclosed membrane structures (PEMS) were isolated as previously described (66 (link)). Briefly, parasite cultures were routinely maintained at <10% parasitemia and 2% hematocrit. Following two rounds of synchronization by sorbitol lysis, the rings were allowed to develop into mature trophozoites and were isolated on a 65% Percoll (Sigma) density gradient. Thereafter, a cysteine protease inhibitor, trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64; Sigma) was added for 8 h to allow growth of late-stage pigmented trophozoites into schizonts without rupture. The treated cultures were pelleted by low-speed centrifugation, resuspended in PBS at 1.8 × 10⁶ schizonts/ml, and immediately frozen at −20°C to lyse the RBCs.

Mouse cortical tissue was homogenized in a 20-fold volume of RIPA buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1% NP-40, 10 mM trans-Epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64) (Sigma), 10 mM Bestatin hydrochloride (Sigma), 10 mM Pepstatin A (Peptide Institute, Osaka, Japan), 10 mM Leupeptin (Peptide Institute), and either 1 mM TME-IAM or 3 mM BM. The homogenate was then centrifuged at 20,380× g at 4 °C for 15 min and incubated at 37 °C for 1 h while rotating. A part of the TME-IAM-treated samples was then diluted with RIPA buffer containing BM (final concentration 3 mM), and further incubated at 37 ℃ for 1 h while rotating. After boiling in SDS sample buffer in the absence of reductants, proteins were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) stain or Western blotting using either Poly-HRP Streptavidin (200,000 dilutions; Thermo Fisher Scientific, Waltham, MA, USA) or rat anti-TME-IAM antiserum (1:3000 dilution). The procedures for signal detection and band intensity quantification were identical to those described in Section 2.7. Protein persulfides were estimated by semi-quantitative analysis of the band intensities of samples treated with both TME-IAM and BM or only BM. The uncropped Western blotting images are shown in the original Western blotting Figure S2.