Abstract
Horse leukocyte elastase inhibitor rapidly forms stable, equimolar complexes with both human leukocyte elastase and cathepsin G, porcine pancreatic elastase, and bovine alpha-chymotrypsin. Formation of the inhibitor-pancreatic elastase complex results in peptide bond cleavage at the reactive site of the inhibitor so that a small peptide fragment representing the carboxyl-terminal sequence of the inhibitor is released. Sequence analysis of both this peptide, as well as that of an overlapping peptide obtained by enzymatic inactivation of native inhibitor with either Staphylococcus aureus metalloproteinase, Pseudomonas aeruginosa elastase, or cathepsin B, yields data which indicate that the reactive site encompasses a P1-P1' Ala-Met sequence. However, unlike the human endothelial plasminogen activator inhibitor, which also has a Met residue in the P1' position, oxidation of the horse inhibitor only slightly reduces its association rate constant with either of the elastolytic enzymes tested or with chymotrypsin. Comparison of the amino acid sequence at or near the reactive site of the horse inhibitor (P2-P18') with members of the serpin superfamily of proteinase inhibitors indicates that it not only belongs in this class but also represents the first example of a functionally active intracellular serpin.
Highlights
Horse leukocyteelastase inhibitor rapidly formssta- The mechanism by which plasma-derived serpins function ble, equimolar complexeswith both human leukocyte is not completely understood
On the basis of both physical and chemical properties, stable equimolar complexes with chymotrypsin, porcine panserine proteinase inhibitors may be divided into two groups creatic elastase, human and horse neutrophil elastase, and which include ( a ) low molecular weight, reversible inhibitors human cathepsin G [19,20,21], suggesting that it might be a reacting according to themechanisms described by Laskowski member of the serpin family
We report the andKat0 [1] and ( b ) high molecular weight, irreversible amino acid sequence at the reactive site of this inhibitor, inhibitors forming equimolar complexes which cannot be dis- results which confirm the fact that thehorse leukocyte cytosol sociated by either low pH or denaturingconditions [2]
Summary
Been classified as members of a superfamily of inhibitors referred to asserpins [3]. The amino acid sequence of at least. Methoxysuccinyl-Ala-Ala-Pro-Val-NSAu,c-Ala-Ala-Pro-Phe-NA, Suc-Ala-Ala-Ala-NA, &isopropyl fluorophosphate, N-chlorosuccinimide, 5,5’-dithiobis(2-nitrobenzoicacid), bovine a-chymotrypsin, and porcine pancreatic elastase were from Sigma. Staphylococcus aureus metalloproteinase and human leukocyte elastase were isolated. Lung and Blood Institute and by Grant 04.01.2.10 from the Polish Academy of Science. Pseudomonas aerugimsu elastase and Ac-Ala-Ala-NHN(CH&OONp were a kind gift of Dr J. Powers (Georgia Institute of Technology, Atlanta, GA). The abbreviations used are: HLEI, horse leukocyte elastase inhibitor; SUC-s, uccinyl-; NaDodSO,, sodium dodecyl sulfate. Chromatography on Gly-Phe-glycinal semicarbazide linked to Sepharose 4B, a kind gift from Dr D. H. Rich (University of Wisconsin, Madison) [24].The elastase inhibitorfrom equine leukocytes (HLEI) was prepared by the method of Dubin [17]
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