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Background: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern., Methods: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy., Results: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation., Conclusions: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein., General Significance: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development., (© 2013.)

The combinatorial approach offered by phage display has proved to be powerful in obtaining novel variants of canonical inhibitors of serine proteinases that show new binding patterns. We applied this strategy to search for variants of basic pancreatic trypsin inhibitor (BPTI) that would be strong inhibitors of two serine proteinases: bovine alpha-chymotrypsin and porcine pancreatic elastase. BPTI only moderately inhibits the first and does not inhibit the second enzyme. A representative library of 3.2 x 10(4) BPTI variants, randomized at P(1), P(1)', P(2)' and P(3)' positions of the proteinase binding loop, was displayed on the surface of phage M13. After four to five rounds of selection on the target proteinase consensus sequences of the inhibitor binding loop were obtained. In both cases, the variants selected differed from BPTI at two to four positions, with a strong preference for selection of hydrophobic residues. Nevertheless, five of these variants expressed in a free form appeared to be correctly folded, stable proteins, and did not aggregate during thermal denaturation. The midpoints of the thermal unfolding curves of these variants were lowered by 5-20 degrees C as compared to BPTI. The expressed variants proved to be new potent inhibitors of the target enzymes with association constants up to 6.9 x 10(9) M(-1) and 3.7 x 10(10) M(-1) for elastase and chymotrypsin, respectively. Thus, the inhibitory properties of BPTI were improved by as much as 7 x 10(6)-fold towards elastase and 420-fold towards chymotrypsin.

A soluble angiotensin (Ang) II-generating enzyme has been purified to homogeneity from the rat mesenteric arterial bed (MAB) perfusate by a combination of gel filtration and affinity chromatographies. The enzyme is a glycoprotein of 28.5 kDa (SDS-PAGE), whose N-terminal sequence is identical with that of the rat pancreatic elastase-2; therefore the enzyme will henceforth be referred to as rat MAB elastase-2. When Ang I was used as the substrate, the enzyme specifically released Ang II and the dipeptide His-Leu (Km=36 microM; Kcat=1530 min-1). The catalytic efficiency (Kcat/Km=42.5 min-1 microM-1) of this reaction was comparable to those of other known Ang I-converting enzymes. The proteolytic specificity of the purified enzyme toward mellitin, oxidized insulin B chain, somatostatin-14 and renin substrate tetradecapeptide suggested that the enzyme-substrate interaction was defined by an extended substrate binding site, typical of elastases-2 of pancreatic origin. According to the sensitivity of the rat MAB elastase-2 to various inhibitors this enzyme could be described as a member of the chymostatin-sensitive group of Ang II-forming serine proteases. The localization and biochemical properties of this enzyme suggest that it might play a role in the regional control of vascular tonus.

A Kazal-type elastase inhibitor was purified by trichloroacetic acid precipitation of sheep lung lavage fluid followed by chymotrypsin affinity and gel-filtration chromatography of the supernatant. Sheep lung elastase inhibitor (SLEI) is glycosylated. Laser desorption mass spectrometry indicated that SLEI has a molecular mass of 16.8-17.3 kDa. Partial protein sequence of SLEI and of a peptide derived from SLEI showed 31-52% and 51-66% homology at the N-terminus and at the inhibitory site respectively with Kazal-type double-headed proteinase inhibitors (bikazins). SLEI inhibited human leukocyte elastase and porcine pancreatic elastase but not human cathepsin G. It was inactivated by chloramine-T and reactivated when incubated with methionine sulfoxide peptide reductase and dithiothreitol, indicating the presence of a methionine at the active site. The concentration of SLEI in bronchoalveolar lavage fluid (BALF) and lung lymph was 0.28 microM (0.23-0.49); 0.24 microM (0.20-0.31) (median, (range), n = 5), respectively and was undetectable in plasma (< 0.03 microM) suggesting that SLEI is produced in the lung. The median molar ratios of SLEI to alpha1-proteinase inhibitor in BALF and lung lymph were 3.2 to 1 and 0.017 to 1, respectively. These results indicate that SLEI probably makes an important contribution to antielastase defence in epithelial lining liquid.

Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography. Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a M(r) greater than about 2000-3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a M(r) of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity.

Upon activation, human polymorphonuclear neutrophils (PMN) release two serine proteinases, cathepsin G (Cat.G) and elastase (HLE), which in turn synergize to activate nearby platelets. We looked for the inhibitory effect of plasma and the involvement of alpha 1 antichymotrypsin (alpha 1 ACT) and alpha 1 antitrypsin (alpha 1 AT), on this cell-to-cell cooperation. It was observed that inhibition by plasma of PMN-mediated platelet activation was rather correlated with an effect on HLE (r = 0.95) than on Cat.G (r = 0.65) enzymatic activity. Purified alpha 1 AT suppressed in a concentration-dependent manner HLE activity present in the supernatant of activated PMN. When HLE was fully blocked, alpha 1 AT started to inhibit Cat.G activity. By contrast and as expected, purified alpha 1 ACT inhibited only Cat.G activity. Using specific blocking polyclonal antibodies against alpha 1 AT and alpha 1 ACT, it was demonstrated that the inhibitory effect of plasma vs. HLE was entirely mediated by alpha 1 AT. By contrast, blockade of Cat.G activity was only partly due to plasma alpha 1 ACT and around 50% was attributable to alpha 1 AT. When plasma from patients with an acute inflammatory state was used in place of plasma from normal subjects, the inhibitory effect was more pronounced, while plasma depleted in alpha 1 AT and alpha 1 ACT was less effective. These data indicate a predominant role of alpha 1 AT in the inhibition by plasma of the PMN-mediated platelet activation.

A series of dihydrouracil derivatives has been synthesized and investigated for their in vitro inhibitory activity toward human leukocyte elastase (HLE) and cathepsin G (Cath G). Alkyl [sulfonyl(oxy)] uracils 1-2 were found to be efficient, time-dependent inhibitors of elastase (kobs/[I] M-1 s-1 values ranged between 480 and 8110). These compounds formed acyl enzymes that exhibited variable hydrolytic stability which appeared to be dependent on the nature of the R1 group (believed to be accommodated at the primary specificity site, S1). The acyl enzymes formed with cathepsin G deacylated rapidly, leading to a significant regain of enzymatic activity. In sharp contrast, the corresponding phosphorus compounds 3-4 were found to be potent, time-dependent irreversible inhibitors of HLE. Furthermore, the results of the structure-activity relationship studies suggest that the binding modes of compounds 1-2 and 3-4 may be different.

Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.

alpha-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of alpha-crystallin, alpha A and alpha B, also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both alpha A-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.

A series of 3-(alkylthio)-N-hydroxysuccinimide derivatives was synthesized and their inhibitory activity towards human leukocyte elastase (HLE) was investigated. The interaction of the compounds having a 3-alkylthioether side chain (compounds 1 and 2) with HLE was found to involve rapid acylation of the enzyme, followed by total regain of enzymatic activity within 3 h. Interestingly, compounds 3-8, having an oxidized thioether side chain, were found to be highly effective, time-dependent, irreversible inhibitors of the enzyme. The k(obs)/I values for compounds 3-8 ranged between 890 and 24,000 M-1 s-1. These findings demonstrate that, unlike the physiological inhibitor of HLE (alpha-1-proteinase inhibitor), which is inactivated upon oxidation, low-molecular-weight compounds retain and/or show enhanced inhibitory activity towards HLE upon oxidation of the thioether side chain and lay the groundwork for the development of compounds that embody proteinase inhibitory and antioxidant activity.

An intestinal elastase was purified from Atlantic cod (Gadus morhua) of apparent molecular mass 24.8 kDa as determined by SDS-PAGE and with isoelectric point above pI 9.3. Heat stability and stability towards acidic pH was reduced in the cod enzyme as compared with porcine intestinal elastase. N-terminal amino-acid sequence analysis of cod elastase showed considerable similarity with porcine elastase. The cod enzyme was less sensitive to phenylmethylsulfonyl fluoride inhibition than porcine elastase, but sensitivity towards other inhibitors was similar. Kinetic properties were examined using the substrate Suc-Ala-Ala-Ala-p-nitroanilide and the cod enzyme was found to have more than 2-times turnover rate (kcat) as compared with the porcine enzyme, and slightly higher Km values. Thus, the catalytic efficiency (kcat/Km) of Atlantic cod elastase was about 2-times higher than observed with porcine elastase, which indicates an adaptive response towards the low temperature environmental in which the cod lives. Substrate specificity was studied by digestion of oxidized B-chain of insulin and by using synthetic substrates. Digestion was most rapid at the carbonyl side of alanine residues, but also occurred at valine and leucine residues.

The role of proteases and reactive oxygen species (ROS) in polymorphonuclear neutrophil (PMN) induced cartilage degradation in vitro were studied. ONO-5046, a novel synthetic elastase inhibitor, significantly and dose dependently protected cartilage from degradation induced by PMNs stimulated with phorbol myristate acetate (PMA), opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine plus cytochalasin-B, or A-23187. The degradation by PMA-stimulated PMNs was unaffected by protease inhibitors which lack anti-elastase activity. However, the hydrogen peroxide (H2O2) reducing agent catalase afforded significant protection. Measurement of elastase activity following PMN activation by PMA showed that antioxidants which reduce H2O2 and/or hypochlorous acid decreased elastase activity. Thus, it is suggested that an indirect interaction between ROS and elastase activity may exist in PMN induced cartilage degradation. Furthermore, the possible implication of an endogenous elastase inhibitor(s) is discussed.

The effect of glucose/Cu2+ incubation on (a) pure methionine oxidation, (b) the oxidation of active-site methionine in alpha 1-proteinase inhibitor (alpha 1PI) and (c) the resulting activity and structural changes of this inhibitor was investigated. While no methionine was oxidized during a 24 day, 37 degrees C incubation with 0.01 M EDTA and 100 mM glucose, 64.2% oxidation occurred in 6 days when 0.01 mM Cu2+ was added to the 100 mM glucose. The first-order rate constant for oxidation in 10 mM glucose, 0.01 mM Cu2+ was 0.0218 day-1. Oxidation was inhibited by catalase, but accelerated by ascorbate ion. The active-site methionyl residue of alpha 1PI was oxidized 71.3% after a 4 day incubation in 100 mM glucose, 0.01 mM Cu2+ (pH 7.45), 0.1 M phosphate buffer. The elastase and trypsin inhibiting activities were lowered to 3.1 and 1.5% of control samples during this incubation. The inclusion of 1 mM DETAPAC, a transition metal chelator, resulted in a 98 + % retention of activity. Intrinsic fluorescence (350 nm excitation, 415 nm emission) of alpha 1PI increased 576% over control for the sample incubated in 100 mM glucose, 0.01 mM Cu2+ and SDS-PAGE revealed protein fragment molecular weights of 44.4 and 39.8 kDa. These studies suggest that both methionine oxidation and free radical induced fragmentation contribute to loss of alpha 1PI activity during glucose/Cu2+ incubations.

Unlike human neutrophil elastase or porcine and rat pancreatic elastases, human pancreatic elastase is rapidly inhibited by oxidized alpha 1-proteinase inhibitor. The second-order association-rate constant for the reaction of the oxidized inhibitor with this enzyme (kass = 10(5) M-1 s-1) is only 8-fold lower than that measured with native alpha 1-proteinase inhibitor. Elastase releases faster from its complex with the oxidized inhibitor (t1/2 approximately 0.7 days) than from its complex with the native inhibitor (t1/2 approximately 5 days). Oxidized alpha 1-proteinase inhibitor is as efficient as the native inhibitor in inhibiting the elastolytic activity of elastase. Oxidized alpha 1-proteinase inhibitor may thus be considered as a physiological inhibitor of human pancreatic elastase which may prevent degradation of blood vessel elastin during acute hemorrhagic pancreatis.

Human plasma prekallikrein, precursor of the bradykinin-generating enzyme, was activated in a purified system under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase which is a tissue-destructive metalloproteinase. Compared with that, Pseudomonas aeruginosa alkaline proteinase poorly activated it with a rate as low as less than one-twentieth of that of elastase. The activation by elastase was blocked with a specific inhibitor of elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2 (10 microM). Generation of kallikrein-like amidolytic activity was also observed in plasma deficient in Hageman factor by treatment with elastase, but was not in plasma deficient in prekallikrein. The kallikrein-like activity generated in Hageman factor deficient plasma as well as the generation process itself was indeed inhibited by anti-human prekallikrein goat antibody. These results suggest that the pathological activation of the kallikrein-kinin system might occur under certain clinical conditions in pseudomonal infections.

Recently we reported a preliminary characterization of anti-elastase activity which is found in cultured keratinocytes and in epidermis from psoriasis patients, but not in normal human epidermis. Here we present evidence that this inhibitory activity is derived from a cationic protein with a molecular mass of 18 kDa. In psoriatic scales the inhibitor is mainly present as a biologically active 11 kDa fragment. Inhibition of human leukocyte elastase is strong (Ki = 2 x 10(11) M) and fast (kon = 10(7) M-1.s-1). Using chromatofocusing, affinity chromatography and gel-permeation FPLC, the 11 kDa fragment was purified from psoriatic scales. This preparation was reduced and carboxymethylated, blotted onto poly(vinylidene difluoride) membrane and subjected to N-terminal gas-phase sequencing. Within a stretch of 16 amino acids a 40% homology was found with the active site of antileukoproteinase (ALP) a known serine proteinase inhibitor present in mucous secretions. We therefore propose the acronym SKALP (skin-derived antileukoproteinase) as a name for this elastase inhibitor.

3-Benzyl-6-chloromethyl-3,4-dihydrocoumarin inhibits human leucocyte elastase (HLE) and porcine pancreatic elastase (PPE) through a mechanism-based process characterized by the following apparent enzyme-inhibitor dissociation constants, Ki, and limiting inactivation rate constants k2: 200 microM (HLE), 69 microM (PPE) and 5.10(-2) s-1 (HLE), 17.7.10(-2) s-1 (PPE) at pH 8.0, 37 degrees C. Bis(4-acyloxyphenyl)methane derivatives with a benzylic halogen as potential leaving group have also been synthesized and studied. They transiently inactivate PPE and HLE through the formation of an acyl-enzyme.

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.

An enzyme with proteolytic activity has been isolated from the subcellular granules of rabbit polymorphonuclear leucocytes. Purification of the enzyme involved extraction of the granule membranes with 0.01 M sodium phosphate buffer, pH 7.4, containing 1 M NaCl and 0.1% Triton X-100, followed by gel exclusion chromatography on Sephadex G-75. The enzyme hydrolysed p-nitrophenol-N-tert-butyloxylcarbonyl-L-alaninate, a synthetic substrate for elastase, but failed to hydrolyse elastin. The enzyme also hydrolysed azo-albumin with a pH optimum between 7.5 and 8.5. Inhibition studies indicated that the enzyme was a serine proteinase (EC 3.4.21.-) and it was found to have an apparent molecular weight of 25 000 by polyacrylamide gel electrophoresis. The purified enzyme behaved as a single on SDS-polyacrylamide gel electrophoresis, had a single isolectric point at pH 5.9, yet showed multiple components on centrifugation.

Pre-alpha 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of alpha 1-proteinase inhibitor and alpha 1-beta 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free alpha 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre-alpha 1-elastase inhibitor and antibodies to alpha 2-beta 1-glycoprotein as well as antibodies to alpha 1-proteinase inhibitor. Pre-alpha 2-elastase inhibitor could be dissociated into its parent components by dithiothreitol. After dissociation, the alpha 1-proteinase inhibitor portion retained its biological activity.

An inhibitor of serine proteinases from human articular cartilage was purified to homogeneity by sequential ultrafiltration and ion exchange chromatography on CM-Sephadex C-50. The apparent molecular weight of the cationic glycoprotein (pI greater than 10) was determined to be 16.5 X 10(3) by SDS gel electrophoresis. The inhibitor blocked the activity of leukocyte elastase, cathepsin G and trypsin but not leukocyte collagenase. In kinetic studies for the interactions with leukocyte elastase a firm enzyme-inhibitor binding was obtained. Amino acid analyses did not reveal homologies with other serine proteinase inhibitors already purified from human tissues.

The properties and subcellular localization of the elastase-like activities of smooth muscle cells cultured from pig aortas have been investigated. Homogenates of the cells hydrolysed N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, a synthetic substrate for elastases, with a distinct pH optimum of 8.2 and hydrolysed insoluble elastin with a distinct pH optimum of 8.5. Both enzyme activities were directly proportional to the concentration of homogenate in the assay mixture. The activities toward both substrates were inhibited by phenylmethylsulphonyl fluoride and were therefore probably due to a serine peptidase(s). The activities were also inhibited by EDTA and, in a dose-related manner, by alpha 1-antiprotease. Pepstatin, which inhibits cathepsin D, and leupeptin, which inhibits cathepsin B, did not significantly inhibit the elastase-like activities in these cells. The cells were homogenized and a post-nuclear supernatant subjected to sucrose density gradient centrifugation. The distribution of elastase-like activity toward both substrates was similar to that of the plasma membrane marker 5'-nucleotidase, and distinct from those of marker enzymes for the other organelles. Cells were also homogenized with digitonin, which selectively increases the equilibrium density of the plasma membrane. The equilibrium densities of both 5'-nucleotidase and of the elastase-like activities were increased considerably, confirming the plasma membrane localization of the elastase-like activities. The subcellular localization of the elastase-like activities of arterial smooth muscle cells is therefore consistent with a role for them in the degradation of elastin in the normal arterial wall and in atherosclerotic lesions.

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.

The inactivation of human pancreatic elastase 2 (EC 3.4.21.11) by a series of peptide chloromethyl ketones has been investigated. Among a series of compounds with the structure X-Ala-Ala-Pro-Y-CH2Cl (where X=acetyl-, succinyl-, methylsuccinyl-, or H-), the kinetic parametrs for inhibition of elastas 2 depend markedly on the amino acid (Y) in the P1 position. Succinyl-Ala-Ala-Pro-Leu-CH2Cl was found to be an extremely effective inhibitor of human elastase 2, qith a first-order rate constant for covalent bond formation (k3) of 0.033s-1 and a dissociation constant, Ki, for the enzyme inhibitor complex of 7.4 . 10(-7) M. The second-order rate constant k3/Ki for inhibition of elastase 2 by the analogous compound containing a free amino group in place of the succinyl moiety is 150 times lower than that found for the succinyl or acetyl derivative, suggesting that the presence of a positive charge at this position reduces the proper binding of the inhibitor to the enzyme.

Chick plasma contains inhibitor(s) against trypsin and elastase which also appear to retard the degradation of tropoelastin by arterial tissue extracts. Chick aorta extracts also contain similar inhibitors against elastase and trypsin. Both levels of the plasma inhibitor(s) and inhibitor(s) extracted from thoracic aorta increase during early stages of growth and maturation. There is a three- to four-fold increase in the levels of the inhibitor(s) in chick plasma and aorta between one to four weeks after hatching. Of particular interest are the observations that the presence of the inhibitor(s) retards the conversion of soluble elastin (tropoelastin) to smaller elastin peptides. Subsequently, it is speculated that in addition to other vital roles, such proteinase inhibitors may also act in regulating elastogenesis and elastin fiber formation.

The actions and availability of human neutrophil elastase and its protein inhibitor, Eglin, when co-incubated with macrophages were investigated. Eglin did not induce radical production by mouse peritoneal macrophages; nor were specific binding sites for Eglin detected on these cells. Mouse peritoneal macrophages could inactivate both elastase and Eglin extensively, when these targets were used at concentrations appropriate to the extravascular fluids. Two methods were used for assessing such inactivation: one, as in previous literature, only took account of molecules remaining in the supernatant after interaction with the cells; the other (lacking from most previous studies) took into account all target molecules, including those associated with the cells. From an analysis of both types of experiment, it was shown that the cell-derived inactivators were stable products, whose quantity was not significantly influenced by the induction of a macrophage oxidative burst and its associated free radicals. They were probably mainly proteinases and proteinase inhibitors. Thus, mouse peritoneal macrophages restrict the activity of proteinases and inhibitors by means of stable molecules, such as proteins. Other mononuclear phagocytes may use free radicals and oxidants more extensively in this respect.

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.

A new and probably unique elastase inhibitor of horse serum was identified, purified to homogeneity and called pre-alpha 2-elastase inhibitor of the horse. Electrophoretically it migrated immediately in front of the alpha 2 position. Its molecular weight was 188 000 by pore limit polyacrylamide gel electrophoresis and 225 000 by Sephadex G-200 gel filtration. The inhibitor was composed of at least two non-identical polypeptide chains of Mr 68 400 and 87 600. A banding pattern of restricted heterogeneity focused between pH 4.9 and 5.2 was revealed by isoelectric focusing. Of 13 animal, microbial and plant proteinases, horse pre-alpha 2-elastase inhibitor inhibited only pancreatic elastase and trypsin efficiently. Chymotrypsin was inhibited only in traces. No analogy between the elastase inhibitor and the known human serum inhibitors could be found with respect to immunological and biochemical criteria.

Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.

The ability of elastatinal and chymostatin, protease inhibitors of microbial origin, to inhibit human leucocyte proteases (EC 3.4.-) was studied. Elastatinal and chymostatin are capable of inhibiting the pancreatic enzymes elastase and chymotrypsin, respectively. It was found in these studies, with synthetic substrates, that elastatinal is a much weaker inhibitor of human leucocyte elastase than it is of porcine pancreatic elastase. Elastatinal caused no inhibition of the activity of human leucocyte chymotrypsin-like protease. Chymostatin was found to be a powerful inhibitor of human leucocyte chymotrypsin-like protease. Its affinity to the leucocyte protease was higher than its affinity to bovine pancreatic alpha-chymotrypsin. Chymsotatin had a weak inhibitory effect on the activity of human leucocyte elastase. Studies were also carried out on the ability of chymostatin to inhibit the release of 35SO2-4 from rabbit articular cartilage by human leucocyte chymotrypsin-like protease. Preincubation of the chymostatin with the protease before the latter was added to the 35SO2-4 -labeled cartilage caused inhibition of proteolysis as measured by 35SO2-4 release. Preincubation of chymostatin with 35SO2-4 -labeled cartilage prior to addition of the human chymotrypsin-like protease to the tissue also inhibited 35SO2-4 release. However, in the case of preincubation of cartilage with alpha1 -antitrypsin there was no such inhibition. It therefore appeared that chymostatin, unlike alpha1 -antitrypsin, was capable of penetrating the cartilage matrix and exerting its inhibitory effect upon the human leucocyte chymotrypsin-like protease that was subsequently added to the tissue.

The lathyrogens, beta-aminopropionitrile and alpha-aminoacetonitrile inhibit both the esterolytic and proteolytic activity of trypsin at a concentration of 100 mM. Lineweaver-Burk plots demonstrate that inhibition is competitive, with alpha-aminoacetonitrile being the more potent inhibitor. The enzyme associated with and capable of digesting tropoelastin is inhibited by both lathyrogens when tested against its natural substrate, tropoelastin. Administration of alpha-aminoacetonitrile-HCl to the diet of young chicks (0.1% w/w) resulted in a 62% increase in the yield of tropoelastin and significant reduction in fatality as compared to beta-aminopropionitrile-fumarate.

The rate constant for the association between human leukocyte elastase (EC 3.4.21.11) and human bronchial inhibitor has been determined by competition experiments with alpha 1-proteinase inhibitor. This constant (1.1.10(7) M(-1) . s(-1)) is 6-times lower than that for the association of leukocyte elastase and alpha 1-proteinase inhibitor. The latter inhibitor is able to dissociate the leukocyte elastase-bronchial inhibitor complex with a rate constant 1.3.10(-4) s-1. The equilibrium dissociation constant Ki of the complex is 1.2.10(-11) M. The physiopathological significance of these constants is discussed.

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.

The mode of inhibition by alpha-1-antitrypsin of chymotrypsin, trypsin and pancreatic elastase was examined by a kinetic method. All three enzymes were completely bound to alpha-1-antitrypsin; therefore, the dissociation constant of the enzyme-inhibitor complex was too low to measure with these methods. The dissociation constants for the three enzyme-inhibitor complexes were estimated to be less than 5 - 10 minus 9 M. However, alpha-1-antitrypsin could be specifically displaced from Sepharose-bound elastase with an irreversible inhibitor of that enzyme. Additional experiments showed that dioxane, 20% (v/v), blocked 100% of the inhibition of elastase, 16% of the inhibition of trypsin, and 0% of the inhibition of chymotrypsin. The effect was reversed by diluting the dioxane to 1% (v/v). These findings indicated that alpha-1-antitrypsin was tightly bound to the three enzymes studied but did not allow discrimination as to the nature of the inhibition. However, the competitive mode of inhibition was suggested by the displacement of alpha-1-antitrypsin from elastase by an irreversible inhibitor of the enzyme that bound covalently at the enzyme-active site. The variable susceptibility of the enzyme to blockage of the inhibition by low concentrations of p-dioxane suggested that hydrophobic bonds may be important in the interaction with alpha-1-antitrypsin.

The reaction of seven N-peptidyl-O-acyl hydroxylamines with serine proteinases exhibiting different substrate specificity has been investigated. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the enzymes may be achieved. Enzyme-catalyzed turnover of inhibitor to products was detected during reaction of proline-specific endopeptidase with N-Boc-Ala-Pro-O-(4-nitrobenzoyl)hydroxylamine.

A plasma alpha 1-proteinase inhibitor was purified, for the first time, from the microsomal fraction of normal human liver by combined procedures of deoxycholate extraction, (NH4)2-SO4 precipitation, gel filtration and immunoadsorbent column chromatographies. The final yield of the material was approx. 15 mg/300 g liver. The preparation was shown to be homogeneous according to polyacrylamide gel electrophoretic and immunological criteria. The estimated molecular weight of the glycoprotein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was 53 000, a value comparable to that reported for plasma alpha 1-proteinase inhibitor. The liver material was, however, devoid of trypsin-, chymotrypsin- or elastase-inhibitory activity. The loss of activity could be due to deoxycholate treatment during the extraction procedure. SDS-polyacrylamide gel electrophoresis of reduced (beta-mercaptoethanol) and alkylated (iodoacetamide) preparation revealed no change in mobility suggesting the presence of only polypeptide chain. The amino acid and carbohydrate compositions of the microsomal material were in close agreement with values reported in the literatures for the corresponding glycoprotein purified from the plasma. The liver microsomal alpha 1-proteinase inhibitor has a single amino terminal residue, namely, glutamic acid, as determined by the dansyl procedure, and a single carboxyl terminal residue, lysine as shown by the carboxypeptidase digestion method. These terminal amino acids are the same as those found in alpha 1-proteinase inhibitor purified from normal human plasma.

Human mucus proteinase inhibitor is a fast-acting inhibitor of human leucocyte elastase (EC 3.4.21.37) and forms a stable, complex with this enzyme. At physiological ionic strength and temperature and in the presence of 10 mg/ml albumin, the kinetic constants characterizing the interaction between elastase and the non-degraded inhibitor are: kass = 6.4.10(6) M-1.s-1, kdiss = 2.3.10(-3) s-1, Ki = 3.10(-10) M. The partially degraded inhibitor isolated by chymotrypsin-Sepharose chromatography inhibits elastase with similar efficiency, suggesting that if partial proteolysis of the inhibitor occurs in vivo, the latter may still act as a potent antielastase. Mucus proteinase inhibitor therefore plays a physiological antielastase function in upper respiratory tract secretions, since it inhibits elastase with a delay time of 150 ms and behaves like an irreversible inhibitor.

Mouse peritoneal exudate macrophage elastase can be significantly purified with 60% recovery of the starting activity by affinity chromatography against SDS-treated alpha-elastin covalently linked to agarose beads. The enzyme has an apparent Mr of 26 500 based on SDS-acrylamide gel electrophoresis. Molecular sieving chromatography on Sephadex gel gives a Mr for macrophage elastase of 21 000--28 000. The enzyme is not inhibited by chloromethyl ketone inactivators specific for pancreatic and leukocyte elastase nor by phenylmethylsulfonyl fluoride. Macrophage elastase also does not bind to tritiated diisopropylphosphorofluoridate. The enzyme is inhibited by EDTA and thus appears to be a metallo-protease. Macrophage elastase is resistant to human alpha 1-proteinase inhibitor and to human and mouse alpha 2-macroglobulin. In view of its lack of susceptibility to these endogenous serum proteinase inhibitors, macrophage elastase may play an important role in physiological and pathological remodeling of connective tissues.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.

The extracts of granules isolated from bovine granulocytes show elastase- and chymotrypsin-like activities, as detected with specific synthetic substrates. Extraction of these enzymes depends upon salt concentration. In the course of the present studies a 21-fold purification of the elastase-like enzyme was achieved on a (Ala)3-CH-Sepharose 4B gel. The molecular weight of the enzyme is 33 000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The elastase-like activity is inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, basic pancreatic inhibitor and by heparin at different rates. Elastatinal inhibits the enzyme competitively (Ki = 80 microM). The cytosol of bovine granulocytes contains a protein which strongly inhibits the elastase-like enzyme of the bovine granulocyte (Ki = 0.4 nM) as well as porcine pancreatic elastase (Ki = 11 nM).

At pH 8.0 and 25 degrees C alpha 1-proteinase inhibitor and alpha 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7.10(5) M-1.s-1 and 6.4.10(6) M-1.s-1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of alpha 1-proteinase inhibitor and alpha 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M-1.s-1. In contrast, alpha 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.