Inhibition of serine proteases: activity of 1,3-diazetidine-2,4-diones.

The present work demonstrates that the 1,3-diazetidine-2,4-dione nucleus is effective as a scaffold of serine protease inhibitors. Compound 1 displayed high activity against human cathepsin G and alpha-chymotrypsin (0.39, 0.69 nM). Compound 6 exhibited 0.85 nM inhibition of human chymase. Compound 10 was a selective inhibitor against human neutrophil elastase.     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

Cleaved SLPI, a novel biomarker of chymase activity

Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.     

Isolation of chymase complexed with physiological inhibitor similar to secretory leukocyte protease inhibitor (SLPI) from hamster cheek pouch tissues

A low molecular weight protein complexed with chymase was isolated from hamster cheek pouch tissues. This protein had an apparent molecular mass of about 10 kDa on SDS-PAGE and the N-terminal sequence showed some homology to secretory leukocyte protease inhibitor (SLPI), which is known as the predominant inhibitor of neutrophil elastase and cathepsin G. Remarkably enhanced inhibition of chymase activity was achieved in the presence of heparin, indicating that the functional property was also similar to SLPI. These findings suggest that this SLPI-like protein is a candidate for a physiological inhibitor of chymase.     

Inhibition of human chymase by 2-amino-3,1-benzoxazin-4-ones

A series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chymase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase. Introduction of an aromatic moiety into the 2-substituent resulted in strong inhibition of chymase, cathepsin G, and chymotrypsin. Extension of the N(Me)CH2Ph substituent by one methylene unit was unfavourable to inhibit these proteases. Towards chymase, 2-(N-benzyl-N-methylamino)-4H-3,1-benzoxazin-4-one (32) and 2-(N-benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-4-one (33) were found to exhibit Ki values of 11 and 17 nM, respectively, and form stable acyl-enzymes with half-lives of 53 and 25 min, respectively. Benzoxazinone 33 also inhibited the human chymase-catalyzed formation of angiotensin 11 from angiotensin I.     

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.     

The degradation of human lung elastin by neutrophil proteinases

Human lung elastin has been isolated by both a degradative and nondegradative procedure and the products obtained found to have amino acid compositions comparable to published results. These elastin preparations, when utilized as substrates for various mammalian proteinases, were solubilized by porcine elastase at a rate six times faster than human leukocyte elastase. Leukocyte cathepsin G also solubilized lung elastin but only at 12% of the rate of the leukocyte elastase. In all cases the elastin prepared by nondegradative techniques proved to be the best substrate in these studies. The differences in the rate of digestion of elastin of the two elastolytic proteinases was readily attributed to the specificity differences of each enzyme as judged by carboxyterminal analysis of solubilized elastin peptides. The plasma proteinase inhibitors, alpha-1-proteinase inhibitor and alpha-2-macroglobulin abolished the elastolytic activity of both leukocyte enzymes, while alpha-1-antichymotrypsin specifically inactivated cathespsin G. Two synthetic inhibitors, Me-O-Suc-Ala-Ala-Pro-Val-CH2Cl (for elastase and Z-Gly-Leu-Phe-CH2Cl (for cathepsin G) were equally effective in abolishing the elastolytic activity of the two neutrophil enzymes. However, inhibition of leukocyte elastase by alpha-1-proteinase inhibitor was significantly suppressed if the enzyme was preincubated with elastin prior to addition of the inhibitor.     

Proteolytic enzyme activities in rat peritoneal exudate.

Proteinase activities in rat thioglycollate elicited peritoneal cells and the cell-free supernatant (lavage fluid) were measured by using the following substrates: Suc-Ala-Ala-Pro-Phe-Methyl-Coumarin-Amide (for cathepsin G or chymase), Suc-Ala-Ala-Ala-AMC (for elastase or elastase-like), Z-Arg-Arg-AMC (for cathepsin B), haemoglobin (for cathepsin D) and Ala-AMC (for alanine-aminopeptidase: AAP). The enzyme activities were correlated to the quantitative distribution of various cell types in the exudate from 0 to 192 nd h. In the supernatant all the examined activities showed a higher value at 72nd h. In the cells activity of chymase and AAP proved to be very high at 0 h but after four h the activities were dropped. From this time all enzyme activities started to elevate till the 24th h. At the 96th h only the activity of cathepsin B and AAP had a high value. We conclude that the intracellular activation and secretion of proteolytic enzymes characteristic for the various peritoneal cell types involved in the acute and chronic inflammatory reaction can be followed by activity measurements using enzyme-specific substrates and inhibitors.     

Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart

Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.     

Finding off‐targets,biological pathways,and target diseases for chymase inhibitors via structure‐based systems biology approach

Off‐target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off‐targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off‐target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off‐targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off‐targets into biological pathways and to establish links between pathways and particular adverse effects. Off‐targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off‐targets. These off‐targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. Proteins 2015; 83:1209–1224. © 2014 Wiley Periodicals, Inc.     

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.     

An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center

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.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.     

Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.     

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.     

Human bronchial leucocyte proteinase inhibitor. Rapid isolation and kinetic analysis with human leucocyte proteinases.

Bronchial leucocyte proteinase inhibitor (BLPI) is an 11 000 Mr protein found in human mucous secretions. This inhibitor apparently controls the serine proteinases elastase and cathepsin G, released from extravascular polymorphonuclear leucocytes. A simple, single-step chromatographic procedure for the isolation of BLPI based on its affinity for chymotrypsin was developed. The purified inhibitor was homogeneous by electrophoresis and gel filtration. Amino acid analyses were in close agreement with previous reports, and showed BLPI to be rich in proline and cystine, but lacking histidine. We have further characterized the role of BLPI with respect to human leucocyte elastase and cathepsin G by close examination of the kinetic parameters. Additionally, we have determined the kinetics of association (kon) and dissociation (koff) for BLPI with bovine trypsin and chymotrypsin. Equilibrium dissociation constants (Ki) of 1.87 X 10(-10) M, 4.18 X 10(-9) M, 8.28 X 10(-9) M and 2.63 X 10(-8) M were obtained for human leucocyte elastase, cathepsin G, bovine trypsin and chymotrypsin, respectively. These results are discussed with respect to BLPI's possible function in vivo and its role relative to other inhibitors in bronchial secretions.