Recombinant eglin c is a potent reversible inhibitor of human pancreatic elastase. At pH 7.4 and 25°C, kass = 7.3 x 105 M-1 s-1, kdiSs =-2.7x 10-4 s-' and Ki = 3.7 x 10-10 M. Stopped-flow kinetics indicate that the formation of the stable enzyme-inhibitor complex is not preceded by a fast pre-equilibrium complex or that the latter has a dissociation constant greater than 0.3 uM. The elastase-eglin c complex is much less stable at pH 5.0 and 25°C, where kdiSs = 1.1 x 10-2 s-I and Ki = 7.3 x 10-1 M. At pH 7.4 the activation energy for kass is 43.9 kJ mol-' (10.5 kcal mol-V). The k>s.increases between pH 5.0 and 8.0 and remains essentially constant up to pH 9.0. This pH-dependence could not be described by a simple ionization curve. Both a2-macroglobulin and a.-proteinase inhibitor are able to dissociate the elastase-eglin c complex, as evidenced by measurement of the enzymic activity of a2-macroglobulin-bound elastase or by polyacrylamide-gel electrophoresis of mixtures of a1-proteinase inhibitor and elastase-eglin c complex. The rough estimate of kdi,. obtained with the a2-macroglobulin dissociation experiment (1.6 x 10-4 S-1) was of the same order of magnitude as the constant measured with the progress curve method. Eglin c strongly inhibits the solubilization of human aorta elastin by human pancreatic elastase. The extent of inhibition is the same whether elastase is added to a suspension of elastin and eglin c or whether elastase is preincubated with elastin for 3 min before addition of eglin c. However, the efficiency of the inhibitor sharply decreases if elastase is reacted with elastin for more prolonged periods. INTRODUCTIONEglin c is an 8.1 kDa protein proteinase inhibitor first isolated from the leech Hirudo medicinalis [1] and now produced by genetic engineering as an N-acetyl derivative [2]. This inhibitor is composed of a single polypeptide chain of 70 amino acid residues. It is extremely stable despite the lack of disulphide bridges [3]. Eglin c belongs to the potato inhibitor I family of serineproteinase inhibitors. It potently inhibits chymotrypsin, subtilisin, neutrophil elastase and cathepsin G, forms loose complexes with bovine pancreatic trypsin and pig pancreatic elastase, and does not inhibit plasmin, thrombin and kallikrein [1,4,5]. The effect of eglin c on human pancreatic elastase has not been reported. This enzyme is a 25 kDa serine proteinase synthesized and stored in the pancreas as a proenzyme. It is composed of a single polypeptide chain of 241 amino acid residues, as determined by sequence analysis of the cloned mRNA [6]. Elastase is able to solubilize fibrous elastin at a high rate [7][8][9]. Its postulated pathogenic role in acute pancreatitis [10][11][12] and atherosclerosis [13] is probably related to its elastolytic properties. The bovine pancreatic trypsin inhibitor (aprotinin), which is sometimes used in pancreatitis, does not inhibit elastase [8]. It was therefore ofinterest to see whether recombinant eglin c inhibits elastase and whether the inhibition also takes place in th...
The kinetic investigation of the inhibition of human pancreatic trypsin 1, trypsin 2 and chymotrypsin A by mucus proteinase inhibitor, eglin c and aprotinin reveals that (i) the first protein is a potent inhibitor of chymotrypsin A (kass. = 1.4 x 10(6) M-1.s-1, Ki = 71 pM) but forms loose complexes with trypsin 1 (Ki = 0.5 microM) and trypsin 2 (Ki = 18 nM), (ii) eglin c does not inhibit the two trypsins but forms a tight complex with chymotrypsin A (kass. = 3.3 x 10(6) M-1.s-1, Ki < 0.1 nM) and (iii) aprotinin is a potent inhibitor of trypsin 1 (kass. = 1 x 10(6) M-1.s-1, Ki < 0.2 nM) and trypsin 2 (kass. = 2.4 x 10(5) M-1.s-1, Ki < 1 nM) but forms a loose complex with chymotrypsin A (Ki = 0.17 microM). These data, together with those published previously on human pancreatic elastase, suggest that a cocktail of aprotinin + eglin c might be a better intensive-care drug for acute pancreatitis than aprotinin alone, because it will efficiently inhibit all four human pancreatic proteinases. On the other hand, human gastric juice inactivates mucus proteinase inhibitor by pepsin-mediated cleavage. This indicates that the fraction of mucus proteinase inhibitor that reaches the stomach following aerosol delivery to cystic fibrosis patients does not reach the duodenum in an active form and, therefore, does not aggravate the pancreatic insufficiency of these patients.
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