leupeptins and benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide

To infect definitive or paratenic hosts, metacercariae of Paragonimus westermani should excyst in the host intestine. Optimum conditions for the excystment have been known to be pH 8-9 and a temperature of 40 C. Under these conditions, excystment of P. westermani metacercariae was accelerated in the presence of 1 mM dithiothreitol (DTT). The DTT acceleration was antagonized dose-dependently by cysteine protease inhibitors of L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64, 2-20 microM) or leupeptin (0.1-1 mM), suggesting that certain cysteine proteases of the metacercaria are involved in excystment. Protease activities were detected in excretory-secretory products (ESP) of newly excysted metacercariae. Two distinct proteases were purified by DEAE anion-exchange chromatography of the ESP. While a 27-kDa protease exhibited endodipeptidolytic activity at pH 5-8.5 and remained stable at neutral pH for 3 days, the 28-kDa enzyme was stable at pH 5-7.5, with lower activity at pH 8.5. Both proteases hydrolyzed collagen, fibronectin, and myosin within 1 hr at pH 8. These results suggest that cysteine proteases secreted by P. westermani metacercariae modulate excystment.

Topics: Animals; Chromatography, Ion Exchange; Collagen; Coumarins; Cysteine Endopeptidases; Dipeptides; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Fibronectins; Hydrogen-Ion Concentration; Iodoacetamide; Leucine; Leupeptins; Myosins; Oligopeptides; Pancreatic Elastase; Paragonimus; Temperature; Trypsin

The effects of peptide proteinase inhibitors on globin hydrolysis by cultured malaria parasites were studied. All of the four cysteine proteinase inhibitors evaluated blocked globin hydrolysis, as documented by the development of a morphological abnormality in which parasite food vacuoles filled with undegraded globin and by SDS-PAGE showing that the cysteine proteinase inhibitor-treated parasites accumulated large quantities of globin. The aspartic proteinase inhibitor pepstatin did not block globin hydrolysis by cultured parasites. None of seven antimalarial drugs tested elicited the food vacuole abnormality caused by cysteine proteinase inhibitors, indicating that this morphological alteration was not simply a sign of nonspecific parasite toxicity. Our results indicate that a trophozoite cysteine proteinase is required for initial cleavages of globin by intact malaria parasites.

Topics: Animals; Antimalarials; Chymotrypsin; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Fluorescent Dyes; Globins; Humans; Hydrolysis; Leucine; Leupeptins; Oligopeptides; Pepstatins; Plasmodium falciparum; Vacuoles

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.

Topics: Caseins; Cathepsin B; Cathepsin H; Cathepsin L; Cathepsins; Cell Fractionation; Cells, Cultured; Concanavalin A; Coumarins; Cysteine Endopeptidases; Dipeptides; Endopeptidases; Fibroblasts; Humans; Hydrogen-Ion Concentration; Hymecromone; Leupeptins; Lysosomes; Microsomes; Skin

The activities of Z-Phe-Arg-NMec(ZPA) hydrolase, cathepsin B and cathepsin H and the concentration of endogenous thiol protease inhibitor in fibroblasts from patients with galactosialidosis were found not to be significantly different from those in control fibroblasts. Culture for 5 days with thiol protease inhibitors such as leupeptin, E-64 or Z-Phe-Phe-CHN2 partially restored the beta-galactosidase activity of fibroblasts from patients, but did not affect the beta-galactosidase activity of fibroblasts from control subjects. However, culture with leupeptin, but not other protease inhibitors, increased the ZPA hydrolase and cathepsin B activities of fibroblasts from both patients and controls 2- to 4-fold. Sephadex G-75 chromatography showed that the activity of high molecular weight ZPA hydrolase, which was initially predominant in fibroblasts, decreased markedly during their culture with leupeptin, while the activities of lower molecular weight ZPA hydrolase and cathepsin B increased about 5-fold. These results suggest that high molecular weight ZPA hydrolase, which is presumably cathepsin J, degrades beta-galactosidase, and that the defect in galactosialidosis is impaired protection of beta-galactosidase from degradation.

Topics: beta-Galactosidase; Cathepsin B; Cathepsin H; Cathepsins; Cells, Cultured; Coumarins; Cysteine Endopeptidases; Diazomethane; Dipeptides; Endopeptidases; Fibroblasts; Galactosidases; Humans; Leucine; Leupeptins; Neuraminidase; Protease Inhibitors