Inhibition of Porcine Elastase by Turkey Ovomucoid and Chicken Ovoinhibitor

Abstract

I. Turkey ovomucoid and chicken ovoinhibitor which have been found to be inhibitors of bovine trypsin and a-chymotrypsin can inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase. The formation of the complex between the elastase and each inhibitor could be demonstrated by means of electrophoresis a t p H 6.9. The complexes are formed in molar ratios of one to one. They are stable and no temporary inhibition could be observed.2. The complexes of porcine elastase with turkey ovomucoid and chicken ovoinhibitor inhibit bovine trypsin a t the same molar ratio as free inhibitors. The complexes of trypsin with these inhibitors inhibit elastase as do the free inhibitors. The complex of porcine elastase with turkey ovomucoid does not inhibit bovine chymotrypsin and the complex of chymotrypsin with this inhibitor does not inhibit elastase. The complex of porcine elastase with chicken ovoinhibitor inhibits bovine chymotrypsin but not the alkaline proteinase from Aspergillus sojae. The complex of this inhibitor with bovine chymotrypsin inhibits the porcine elastase while the complex iwith alkaline proteinase does not.It was concluded, therefore, that turkey ovomucoid has two independent binding sites, one for trypsin and the other for chymotrypsin or elastase and that chicken ovoinhibitor has three independent binding sites, one for trypsin, another for chymotrypsin and a third one for either elastase or alkaline proteinase.3. The complexes between porcine elastase and turkey ovomucoid or chicken ovoinhibitor are not adsorbed on elastin whereas elastase is. 4.Kunitz soybean and pancreatic trypsin inhibitors, Birk-Bowman soybean trypsin and chymotrypsin inhibitor, groundnut and h a bean trypsin and chymotrypsin inhibitors do not inhibit the esterolytic, proteolytic and elastolytic activities of porcine elastase, even in weight ratios in excess of 10 : 1.Although the tertiary structure and the catalytic site of porcine elastase are similar to those of bovine trypsin and chymotrypsin, its binding site and substrate specificity are quite different [I, 21. Elastase is not inhibited by most of the natural trypsin and chymotrypsin inhibitors [3]. Several reports described inhibition of porcine elastase by trypsin inhibitors from plants or animals 14-81 but in most cases only the non-specific proteolytic activity was inhibited while the elastolytic activity was almost unaffected An introduction of a highly specific esterase substrate for porcine elastase, N-acetyl-L-alanyl-Lalanyl-L-alanine methyl ester [I21 enhanced our studies on specific inhibition of porcine elastase by natural inhibitors.The present study describes the inhibition of porcine elastase by turkey ovomucoid and chicken ovoinhibitor and partial characterization of their complexes with this enzyme.

Purification and cDNA cloning of a four‐domain Kazal proteinase inhibitor from crayfish blood cells

“…Thus, the first four domains are all potentially active against trypsin, the fifth domain against chymotrypsin and the sixth and seventh domains against chymotrypsin and elastase. However, the intact protein has only two sites for trypsin, two sites for chymotrypsin [30] and one for elastase [31]. Similarly, the three-domain chicken ovomucoid has lysine, arginine and alanine residues at its putative reactive sites [28].…”

Elastase secretion by stimulated macrophages. Characterization and regulation.

“…Unlike these enzymes the macrophage elastase does not attack synthetic substrates such as Ac-(Ala),-4-nitroanilide, or the less specific elastoesterase substrates in a way which releases the chromogenic end groups . It is also relatively resistant to the active site-directed alanyl chloromethyl ketones designed for pancreatic elastase (27) and is unaffected by turkey ovomucoid, inhibitor of both the other elastases (10,28,29) . Although all three types of elastase are serine proteinases sensitive to Dip-F and chicken ovoinhibitor (10,28), the macrophage and pancreatic enzymes differ from the granulocyte elastase in their sensitivity to chelating agents and resistance to soybean trypsin inhibitor.…”

“…It is also relatively resistant to the active site-directed alanyl chloromethyl ketones designed for pancreatic elastase (27) and is unaffected by turkey ovomucoid, inhibitor of both the other elastases (10,28,29) . Although all three types of elastase are serine proteinases sensitive to Dip-F and chicken ovoinhibitor (10,28), the macrophage and pancreatic enzymes differ from the granulocyte elastase in their sensitivity to chelating agents and resistance to soybean trypsin inhibitor. With respect to its limited protein substrate specificity and metal ion requirements, the macrophage elastase resembles the microbial elastases isolated from Flauobacterium elastolyticum (32) and Pseudomonas aeruginosa (33), respectively, the latter being a common contaminant of water supplies and thus, possibly, elastin plates .…”

The significance of the n‐acyl residue of L‐alanyl‐L‐alanyl‐L‐alanine p‐nitroanilide for cleavage by pancreatic elastase

“…The adsorption of elastase on substrate (elastin) has according to Gertler [2] two prerequisites: electrostatic charge and nonpolar residue. We presume that the presence of a negatively charged carboxyl anion In N-acylated p-nitroanilide peptide makes possible electrostatic bonding with the positively charged enzyme groups.…”

“…It is of importance in the pathogenesis of acute pancreatitis, especially in damage to the blood vessels [ 1 ] . It is not inhibited by clinically used protease inhibitors of the Kunitz type [2]. New specific substrates of various types for the estimation of elastolytic activity have been synthesized [3-51.…”