Elastase proteases

Keywords human elastase, protease inhibitors, silicon diol, privileged structure. 

If the yield of viable cells is low be sure that the enzymes are stored cold and dry and that the aliquots of enzyme are stored frozen use more collagenase depending upon tissue type, additional enzymes may be needed, e.g., elastase, protease. 

Effects of proteolytic enzymes on the cohesion of chunks of poultry meat have been studied to determine the mechanism of heat-initiated cohesion (107). All the proteolytic enzymes tested showed a decrease in binding scores in the following order collagenase, ficin, elastase, protease, papain, trypsin, and chymotrypsin. The involvement of stromal proteins in binding is suggested. 

Furthermore, it has become clear recently that even the subminimum inhibitory concentration of 14-membered ring macrolides has the inhibitory effects of biofilm formation and expression of virulence factors (piocyanin, elastase, proteases) of P. aeruginosa (see following section). Thus, an anti-inflammatory agent such as EM should be used as basic treatment in the therapy of chronic airway infection. 

One intensively investigated feature of the inflammatory process in COPD is the release of proteases from neutrophils and monocytic cells that destroy elastin and other components of the interstitial matrix (Table 1). The best studied protease is neutrophil elastase. Independent of its elastolytic activity, neutrophil elastase is a potent secretagogue. More recently matrix metalloproteases (MMP) have received increasing attention, in particular MMP 12 (macrophages elastase). To which extent and how exactly these proteases become activated is not clear at present. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

Further, Wasserman and coworkers developed a direct acylation of stabilized phosphonium ylides by carboxylic acids in presence of the EDCI/DMAP (way c). This last method allows the introduction of a-aminoacid structures into the resulting P-oxo phosphorus ylides [19-25],opening the way to the total synthesis of depsipeptide elastase inhibitors [22,24] or cyclic peptidic protease inhibitor EurystatinA [20]. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

OCi-Antitrypsin is the major serine protease inhibitor of plasma, in particular inhibiting the elastase of neu-... 

Alpha-l-antiprotease (ai-AP) limits tissue damage arising from the actions of the leucocyte protease, elastase (Carrell and Travis, 1985), and there is much evidence available for the oxidative inactivation of this protein by oxygen-derived free-radical species and hypochlorous acid/hypochlorite anion (HOCl/OCP). The mechanism of this inactivation appears to involve the oxidation of a critical methionine residue (Met-358) to its corresponding sulphoxide and methionine sulphoxide has been detected in ai-AP samples isolated from the lungs of cigarette smokers (Carp et al., 1982) and rheumatoid synovial fluids (Wong and Travis, 1980). 

Serine proteases usually show primary specificity (occupation of subsite Si) for positively charged arginine or lysine (trypsin, plasmin, plasminogen activators, thrombin), large hydrophobic side chains of phenylalanine, tyrosine, and tryptophan (chymotrypsin, cathepsin G, chymase, and subtilisin), or small aliphatic side chains (elastases). Nevertheless, there are a large number of variations and in many cases, other subsites like S2 and S3 are more discriminating while maintaining the... 

Finally, coumarin derivatives may act as general inhibitors of serine proteases or as specific inhibitors of human leukocyte elastase, depending on the nature of the substituents, through two distinct mechanisms, suicide substrates (a-chymotrypsin)... 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Human leukocyte elastase is a protease that degrades elastin and other connective tissue components. It is implicated in the pathogenesis of pulmonary emphysema and other inflammatory diseases such as rheumatoid arthritis and cystic fibrosis. Porcine pancreatic elastase has often been used as a model for HLE. Both enzymes have a small primary binding site Si. 

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

Several of the postulated roles for nematode-secreted AChEs assume that they gain access to the intestinal mucosa. Several possibilities exist for transport of parasite AChE across the epithelial cell barrier, such as (i) utilization of existing pathways for receptor-mediated transcytosis (ii) a paracellular route facilitated by parasite-secreted proteases as observed for a bacterial elastase (Azghani et al., 1993) and (iii) increased paracellular permeability resulting from inflammatory events in the mucosa. We consider the latter suggestion most likely, as this has been duplicated by ex vivo perfusion with rat mast cell protease II (Scudamore et al., 1995). Moreover, cholinergic stimulation attenuates epithelial barrier properties to macromolecules in rat ileal crypts (Phillips et al., 1987). 

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