Leupeptins, protease inhibitor

More than 50 endogenous and exogenous inhibitors of the calpains have been described as either transition-state reversible or irreversible inhibitors. The first transition-state inhibitors were the peptide aldehydes (e.g., leupeptin). Using this compound, new ones were synthesized that exhibited improved membrane permeability and calpain specificity (e.g., calpeptin). Other groups of inhibitors have since been discovered a-dicarbonyls (originally developed as serine protease inhibitors), nonpeptide quinolinecarboxamides,... 

As shown in Table I, free HRP is poorly transported across MDCK cells but, when conjugated to a PLL carrier, HRP transport is increased considerably. The existence of a proteolytic compartment involved in the transcytotic digestion of HRP-S-PLL conjugate was further confirmed by the finding that when PLL was replaced by PDL, the transport of HRP was completely abolished (Table I) (8). In addition, when protease inhibitors such as leupeptin were added to the basal medium, the transcytosis of HRP was also significantly decreased (Table I). We have previously reported that the partial degradation of HRP-S-PLL was not inhibited by lysosomotropic amines (<8), indicating that this proteolytic process does not occur in lysosomes. 

Mammalian Cell Protease Inhibitor CocktaiL These should contain AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. (Metal chelators can be added to suppress the activity of calcium ion-dependent proteases such as calpain. Again, one must determine whether the protein or enzyme being purified does not require a divalent metal cofactor for stabihty or activity.)... 

A number of inhibitors of carcinogen- and radiation-induced ceU transformation have been identified. These include protease inhibitors (antipain, leupeptin, soybean tryspin inhibitor), vitamin A analogs (retinoids), vitamin C (ascorbic add), glucocorticoids, and lymphotoxins (Kennedy, 1984). 

A distinctive feature of calpain-3 is the propensity to autolysis the half life of the protein in vitro is less than 30 minutes. Autoproteolysis is prevented by replacing the cys residue of the catalytic site with a ser, but is not affected by cysteine protease inhibitors such as calpastatin, E64 and Leupeptin (Sorimachi et al., 1993). The autocatalytic activity and the absence of effective inhibitors have made the... 

Peptides that display a terminally located aldehyde function in their structure constitute another group of modified peptide enzyme inhibitors. The sequence benzyloxycarbonyl-Pro-Phe-CHO fulfils the known primary and secondary specificity requirements of chymotrypsin and has been found to be a potent reversible inhibitor of this proteolytic enzyme (Walker et al. 1993). Further, protease inhibitors comprising terminally located aldehyde function are antipain, leupeptin, chymostatin, and elastatinal. In addition, also phos-phoramidon, bestatin, puromycin, and amastatin represent modified peptides which can reversibly inhibit enzymes. 

Matsumura, Y. Kusuoka, H. Inoue, M. Hori, M. Kamada. T. Protective effect of the protease inhibitor leupeptin against myocardial stunning. / Cardiovasc. Pharmacol. 22 135-142 1993. 

G proteins are located in different compartments within the cell (Nurnberg and Ahnert-Hilger, 1996). Although most G proteins are found attached to the plasma membrane and intracellular membranes, some are also located within the cytoplasm (Rudolph et al., 1989). Therefore, G proteins in preparations of disrupted cells, or in cell and tissue extracts are also subject to pertussis toxin-mediated ADP-ribosylation. In this case, precautions have to be taken to prevent proteolysis, and protease inhibitors should be included in the buffer (aprotonin, p-aminobenzamidine, leupeptin, phenylmethylsul-fonyl fluoride, or soybean or lima bean trypsin inhibitors) (Carty, 1994). 

PTHrP is unstable in serum and plasma at 4 °C and at room temperature unless collected in the presence of protease inhibitors.A combination of aprotinin, leupeptin, pepstatin, and EDTA provides the greatest protection. In general, specimens should be collected with protease inhibitors and kept on ice. Serum or plasma should be promptly separated from the clot and/or cells and frozen. 

Proteins to be tested were diluted in buffer E (10 mM HEPES-KOH, pH 7.4,100 mM KC1), at five times their final concentration in the assay. Assay volume was 25 qL and assays were performed in 0.5 mL microcentrifuge tubes. Assays used Buffer L (125 mMKCl, 0.5 mMMgCl2, 3.0 mM succinic acid, 3.0 mM glutamic acid, 10 mM HEPES-KOH, pH 7.4, containing 25 qg/mL leupeptin, 25 qg/mL pepstatin, 3 qg/mL aprotinin, 100 mMPMSL, and 10 qM of the caspase inhibitor Boc-Asp-LMK). Protease inhibitors were added immediately to Buffer L prior to its use. 

High-performance size-exclusion chromatography (HPSEC) was used for competition studies. Solubilized HLA-DR1 (0.13 pM) was incubated for 48 h at 37 °C with the N-ter-minally 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled IM-(19-31)-peptide dissolved in 150 mM sodium phosphate, pH 5.5, containing 15 % (v/v) acetonitrile, 0.1 % (w/v) Zwittergent-12 (Calbiochem) and a cocktail of protease inhibitors (0.2 mM PMSF, 5 pM leupeptin, 10 pM pepstatin, and 1 pM chymostatin). Competition assays were performed in a 1.5 pM solution of AMCA-peptide as competitors, different peptides, the peptide library or peptide sublibraries were added in concentrations ranging from... 

Proteases may be promising targets for drug intervention since proteolytic events have been shown to be critical for successful invasion of the red cell by the merozoite (Blackman, 2000 O Donnell and Blackman, 2005). For example, in vitro invasion by falciparum merozoites can be blocked by the serine and cysteine protease inhibitors, chymostatin and leupeptin moreover, these inhibitors act at different steps in the invasion process. Chymostatin inhibited both invasion and rupture of... 

Microorganisms produce a number of antibacterial substances of low molecular weight such as penicillin and streptomycin. In the past it was believed that microorganisms produce antibacterial substances to suppress their competitors. However, in the late 1960 s screening of enzyme inhibitors started, and most of enzyme inhibitors of microbial origin did not show any antibacterial activity. For example, protease inhibitors from Streptomyces, such as leupeptin and antipain, are very specific to enzyme inhibition and show no antibacterial effect. Therefore, it is more likely that these antibiotics and enzyme inhibitors have absolutely no role in the producing strains and that they are only the products of remnant genes that have lost their relevance over the course of evolution. 

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