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• W2022603931 abstract "This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment (30bpDNA), used as a model of DNA, tightly binds cat G (K d, 8.5 nm) but does not react with proteinase 3, α1PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whoseK i is close to K d. ACT and α1PI are slow binding inhibitors of the cat G-30bpDNA complex whose second-order rate constants of inhibition are 2300 m−1 s−1 and 21 m−1 s−1, respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, 30bpDNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by α1PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases. This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment (30bpDNA), used as a model of DNA, tightly binds cat G (K d, 8.5 nm) but does not react with proteinase 3, α1PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whoseK i is close to K d. ACT and α1PI are slow binding inhibitors of the cat G-30bpDNA complex whose second-order rate constants of inhibition are 2300 m−1 s−1 and 21 m−1 s−1, respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, 30bpDNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by α1PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases. neutrophil cathepsin G α1-proteinase inhibitor (α1-antitrypsin) α1-antichymotrypsin mucus proteinase inhibitor (secretory leukoprotease inhibitor) succinyl p-nitroanilide 30-base pair DNA fragment carbobenzoxy-Gly-Leu-Phe-chromethylketone Neutrophils are phagocytic cells recruited at sites of infection and inflammation. Their azurophil granules, which participate in phagocytosis, store a number of hydrolytic enzymes including elastase, cat G,1 and proteinase 3, three serine proteinases whose tertiary structure has been elucidated recently. These 25–30-kDa glycoproteins contain a large number of basic amino acid residues that are responsible for their cationic character. Their specificity is directed against small aliphatic amino acid residues (elastase and proteinase 3) or more bulky ones (cat G).In vitro, these enzymes are able to cleave lung extracellular matrix proteins such as elastin, collagen, fibronectin, and laminin. They also cause extensive lung tissue damage in the animal (1.$$$$$$ ref data missingGoogle Scholar). Ideally, digestion of phagocytosed material should take place within the neutrophil in a phagocytic vacuole filled with the above proteinases. Actually, however, part of the lysomal enzymes reaches the extracellular space as a result of incomplete closure of the phagolysosome or of frustrated phagocytosis of large particules. In addition, neutrophils are short lived cells that release the bulk of their proteinases when they die at sites of inflammation. Tissues are normally protected against these enzymes by a number of proteinase inhibitors. For example, lung secretions contain at least three antiproteinases, namely α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI) (2.Tegner H. Acta Oto-laryngol. 1978; 85: 282-289Crossref PubMed Scopus (57) Google Scholar). The 53-kDa α1PI and the 68-kDa ACT are glycoproteins synthesized in the liver and transported into the lung via the blood circulation. They belong to the serpins, a superfamily of proteins that have developed by divergent evolution over 500 million years. The serpins have a highly conserved secondary structure comprising nine α-helices and three β-sheets with a very flexible reactive site loop. They form denaturant-stable complexes with their target proteinases and behave kinetically like irreversible inhibitors. This irreversible binding is due to the formation of a nonhydrolyzable acyl-enzyme adduct between the serine residue of the enzyme's active site and the P1 residue of the serpin. The P1 residues of α1PI and ACT are Met and Leu, respectively (3.Gettins P.G.W. Patston P.A. Olson S.T. Landes R.G. Serpins: Structure, Function and Biology. Springer, New York1996Google Scholar). α1PI inhibits the three aforementioned proteinases, whereas ACT reacts only with cat G. The second-order rate constants for these serpin-proteinase associations range from 105 to 107m−1 s−1 (1.$$$$$$ ref data missingGoogle Scholar). MPI is a 11.7-kDa basic protein synthesized and secreted by bronchial epithelial cells. It is formed of a single chain of 107 amino acid residues organized in two domains of similar size and architecture. It has a Leu residue at the P1 position of its reactive center. MPI belongs to the class of canonical inhibitors that have a rigid reactive site loop that forms a reversible “lock and key” complex with the target proteinase. This complex is stabilized by a large number of noncovalent bonds, which account for the high enzyme-inhibitor affinity (4.Bode W. Huber R. Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1002) Google Scholar). MPI is a tight binding reversible inhibitor of elastase (K i, 32 pm (5.Cadène M. Boudier C. Daney de Marcillac G. Bieth J.G. J. Biol. Chem. 1995; 270: 13204-13209Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar)). It is less efficient on cat G (K i, 35 nm(6.Boudier C. Cadène M. Bieth J.G. Biochemistry. 1999; 38: 8451-8457Crossref PubMed Scopus (20) Google Scholar)) and does not inhibit proteinase 3 (7.Rao N. Wehner N. Marshall B. Gray W. Gray B. Hoidal J. J. Biol. Chem. 1991; 266: 9540-9548Abstract Full Text PDF PubMed Google Scholar). Because of their ability to cleave lung extracellular matrix proteinsin vitro, neutrophil proteinases are thought to cause tissue destruction in inflammatory lung diseases. This raises, however, the following question: why does proteolysis occur despite the presence of the aforementioned inhibitors? Oxidative inactivation of proteinase inhibitors, transient excess of proteinase over inhibitor, and close neutrophil/matrix contact are conditions that favor proteolysis in the presence of inhibitors (1.$$$$$$ ref data missingGoogle Scholar). Binding of proteinases to DNA released from neutrophils following cell death may also promote proteolysis in an inhibitory environment. We have recently shown that polynucleotides bind neutrophil elastase and impair its inhibition by MPI and α1PI (8.Belorgey D. Bieth J., G. FEBS Lett. 1995; 361: 265-268Crossref PubMed Scopus (34) Google Scholar, 9.Belorgey D. Bieth J.G. Biochemistry. 1998; 37: 16416-16422Crossref PubMed Scopus (27) Google Scholar). Here we study the influence of neutrophil DNA on the inhibition of neutrophil cat G and proteinase 3 by α1PI, ACT, and MPI. To this end, we use a 30-base pair DNA fragment (30bpDNA) as a handy model of DNA. Human neutrophil cat G was isolated and active site titrated as described previously (10.Boudier C. Holle C. Bieth J.G. J. Biol. Chem. 1981; 256: 10256-10258Abstract Full Text PDF PubMed Google Scholar). Human neutrophil proteinase 3 and human plasma ACT were purchased from Athens Research and Technology (Athens, GA). Human recombinant α1PI and MPI were obtained through the courtesy of Dr. H.P. Schnebli, Novartis, Switzerland. α1PI and MPI were active site titrated with elastase (11.Bruch M. Bieth J.G. Biochem. J. 1986; 238: 269-273Crossref PubMed Scopus (54) Google Scholar), whereas ACT was titrated with cat G (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar). Proteinase 3 was titrated with α1PI (7.Rao N. Wehner N. Marshall B. Gray W. Gray B. Hoidal J. J. Biol. Chem. 1991; 266: 9540-9548Abstract Full Text PDF PubMed Google Scholar). Recombinant human pancreatic DNase I (Pulmozyme, Genentech, San Francisco, CA) was obtained from the Pharmacy of the University Hospital of Strasbourg. Thep-nitroanilide and the thiobenzylester substrates came from Bachem (Switzerland) and E.S.P. (Livermore, CA), respectively. Stock solutions of the substrates and of 4,4′-dithiodipyridine (Sigma) were made in dimethylformamide (final concentration, 2% (v/v)). Z-Gly-Leu-Phe CH2Cl and chymostatin came from E.S.P. and Sigma, respectively. All experiments were done at 25 °C in 50 mm Hepes, 150 mm NaCl, pH 7.4, a solution called the buffer. The bronchial secretions from cystic fibrosis patients were used as a source of neutrophil DNA. The viscous fluids were incubated for 3 h at 50 °C with 0.5% SDS, 100 μg/ml proteinase K (Sigma), 20 μg/ml RNase (Sigma), and 5 mm EDTA. DNA was then extracted using phenol:chloroform:isoamylic alcohol (25:24:1) and precipitated with ethanol (13.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The pellet was then dissolved in the buffer (2 mg/ml) and reacted with 290 nm DNase for 90 min at 25 °C. After heating for 5 min at 65 °C, the mixture was chromatographed on a 12 HR 10/30 Superose column (Amersham Pharmacia Biotech), calibrated with the pBr 322 plasmid previously digested with HaeIII (14.Früh H. Kostoulas G. Michel B.A. Baici A. Biol. Chem. Hoppe-Seyler. 1996; 377: 579-586Crossref PubMed Scopus (23) Google Scholar). The fractions containing DNA fragments of about 30 base pairs (M r 20,000) were collected and concentrated. The purity was checked using polyacrylamide gel electrophoresis (Phastsystem, Phast Gel 20 and DNA silverstaining kit from Amersham Pharmacia Biotech). Sepharose-bound 30bpDNA and MPI were prepared using epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The affinity supports were poured into HR 10/10 Amersham Pharmacia Biotech columns and equilibrated with 50 mm Hepes buffer, pH 7.4. cat G, proteinase 3, α1PI, and ACT were dissolved in this buffer at a concentration of 0.2–0.3 mg/ml, loaded on the Sepharose-30bpDNA column and eluted with a linear NaCl gradient in the same buffer. Protein elution was followed at 280 nm.30bpDNA (20 μg/ml) was dissolved in the above buffer and chromatographed on the Sepharose-MPI column. NaCl gradient elution was followed at 260 nm. Fluorescent labeling of cat G was done by reacting 1 mol of cat G with 10 mol of 5-(4, 6-dichlorotriazinyl)aminofluorescein (Molecular Probes, Eugene, OR) in 0.2 m carbonate buffer, pH 8.4. After 15 min of continuous stirring at room temperature, labeled cat G was isolated using a PD-10 gel filtration column (Amersham Pharmacia Biotech) equilibrated with a buffer formed of 50 mm Hepes and 150 mm NaCl, pH 7.4. Constant concentrations of labeled cat G were mixed with increasing concentrations of 30bpDNA, and the fluorescence intensity of the mixtures was determined with a Shimadzu RF 5000 spectrofluorimeter (λex, 495 nm; λem, 514 nm) The data were fitted to Equation 1 by nonlinear regression analysis, which gave the best estimate of K d and its confidence interval. ΔF=([E] o+[D] o+Kd)−([E] o+[D] o+Kd) 2−4[E] o[D] o2[E] oEquation 1 ×ΔFminwhere D stands for 30bpDNA, ΔF and ΔF min are the absolute variations of fluorescence intensity at a given 30bpDNA concentration and at saturating 30bpDNA concentrations, respectively. The inhibition of cat G by30bpDNA was monitored using a synthetic substrate as described in the legend to Fig. 3. The inhibition of 30 nmproteinase 3 by concentrations of 30bpDNA up to 1 μm was assessed using 2 mmmethoxysuccinyl-Lys(2pico)-Ala-Pro-Val-pNA (14.Früh H. Kostoulas G. Michel B.A. Baici A. Biol. Chem. Hoppe-Seyler. 1996; 377: 579-586Crossref PubMed Scopus (23) Google Scholar). The effect of 30bpDNA on the elastolytic activity of these two proteinases was tested using Remazol Brilliant Blue elastin as described previously (8.Belorgey D. Bieth J., G. FEBS Lett. 1995; 361: 265-268Crossref PubMed Scopus (34) Google Scholar). The final concentration of the proteinases was 0.5 μm. One single concentration of30bpDNA (15 μm) was used in replicate assays. Rate constants of inhibition were measured by adding cat G ± 30bpDNA to a mixture of inhibitor + substrate ± 30bpDNA and recording the release of product as a function of time. When the progress curves lasted for more than 10 min, an ordinary spectrophotometer (Uvikon 941, Kontron) was used to record them. When the reaction was terminated in 2 min or less the Uvikon spectrophotometer was equipped with a SFA-11 fast mixing accessory (High-Tech Scientific, Salisbury, UK). When faster reactions were followed, mixing of the reagents and data acquisition were performed with a PQ/SF-53 stopped flow apparatus (High Tech Scientific). All experiments were done under pseudo-first-order conditions, that is [I]o ≥ 10[E]o. The concentration of product at the end of the inhibition experiments was always lower than 5% of the initial substrate concentration so that the latter was virtually not depleted during the inhibition process. Under these two conditions the progress curves describing the enzyme-catalyzed hydrolysis of substrate in the presence of irreversible inhibitors such as ACT, α1PI, or Z-Gly-Leu-Phe-CH2Cl may be described by Equation 2 (15.Bieth J.G. Methods Enzymol. 1995; 248: 59-84Crossref PubMed Scopus (186) Google Scholar,16.Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar), [P]=vzk(1−e−kt)Equation 2 where [P] is the product concentration at any timet, vz is the velocity att = 0, and k is the pseudo-first-order rate constant of inhibition. The progress curves were fitted to Equation 2by nonlinear least square analysis to obtain the best estimates ofk. The variation of k as a function of inhibitor or substrate concentration was analyzed assuming either that the inhibition conforms to a simple bimolecular reaction E +I →kass EI or that it takes place in two-steps. E+I ⇌Ki* EI*→k2 EI.Equation 3 The rate constant of inhibition is given by Equation 4 (one-step inhibition) or Equation 5 (two-step inhibition). k=kass[I] o1+[S] o/KmEquation 4 k=k2[I] o[I] o+Ki*(1+[S] o/Km)Equation 5 Equations 4 and 5 show that a linear or a hyperbolic plot ofk versus [I]owill diagnose one-step or two-step inhibition, respectively. Two-step inhibition does not, however, necessarily yield a hyperbolic dependence of k versus[I]o; if the largest [I]o used is significantly lower than K i*(1 + [S]o/K m), Equation 5 reduces to k =k 2[I]o/K i*(1 + [S]o/K m), a linear equation that resembles Equation 4. The progress curves for the inhibition of 30bpDNA-bound cat G by the reversible inhibitors MPI and chymostatin may be described by Equation 6 (15.Bieth J.G. Methods Enzymol. 1995; 248: 59-84Crossref PubMed Scopus (186) Google Scholar, 16.Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar), [P]=vst+vz−vsk(1−e−kt)Equation 6 where vs is the steady-state velocity, and the other symbols have the same meaning as in Equation 2. The variation of k as a function of inhibition concentration was analyzed assuming one-step inhibition: E+I ⇌kdisskass EIEquation 7 for which k is given by: k=kass[I] o1+[S] o/Km+kdissEquation 8 One volume of a buffered solution of cat G ± 30bpDNA was mixed with an equal volume of a continuously stirred suspension of Remazol Brilliant Blue elastin ± α1PI ±30bpDNA. While stirring was continued at 37 °C, 0.5-ml aliquots were withdrawn after given periods of time and diluted with 0.5 ml of 0.75 m acetate buffer, pH 5.0, to stop the reaction. After centrifugation at 10,000 × g for 15 min, the absorbances were read at 595 nm. The absorbances of appropriate controls were substrated. The buffer was 50 mmHepes, 150 mm NaCl, pH 7.4. The final reactant concentrations were: cat G = α1PI = 1 μm, 30bpDNA = 2 μm, elastin = 3 mg/ml. Fig.1 A shows that at low ionic strength (50 mm Hepes, pH 7.4) Sepharose-30bpDNA binds ACT and cat G but does not significantly bind α1PI. MPI was weakly bound and was eluted with a low NaCl concentration (not shown). Whereas cat G was tightly bound to the affinity support, ACT was eluted at a NaCl concentration lower than that contained in the buffer used for the enzyme kinetic assays, namely 50 mm Hepes, 150 mm NaCl, pH 7.4. The weak binding of ACT to the affinity column is not surprising, because ACT is known as a DNA-binding protein whose binding domain has been identified recently (17.Naidoo N. Cooperman B.S. Wang Z.M. Liu X.Z. Rubin H. J. Biol. Chem. 1995; 270: 14548-14555Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To further confirm the weak binding of MPI to the polynucleotide, we have chromatographed 30bpDNA on a Sepharose-MPI column. Fig.1 B shows that the polynucleotide elutes from this column with a NaCl concentration of about 100 mm. Thus, cat G is the only protein that binds 30bpDNA in the aforementioned buffer. In an attempt to quantitate the cat G-30bpDNA affinity we have measured the effect of polynucleotide concentration on the fluorescence intensity of fluorescently labeled cat G. Fig.2 A shows that this intensity decreases up to a limiting value F min. Fig.2 B is a replot of these data in accordance with Equation 1from which K d, the equilibrium dissociation constant of the cat G-30bpDNA complex, was calculated by nonlinear regression analysis. K d was found to be 8.5 ± 3.2 nm. Reaction of constant amounts of cat G with increasing amounts of 30bpDNA resulted in partial inhibition of the enzymatic activity on Suc-Ala2-Pro-Phe-pNA, whether the enzyme concentration was low (24 nm) or high (300 nm) (Fig. 3). When the inhibition of 24 nm cat G was measured using different substrate concentrations, similar results were obtained (data not shown). Partial inhibition may be analyzed using the scheme adapted from Ref. 18.$$$$$$ ref data missingGoogle Scholar, E+S ⇌Km ES →kcat E+P ++ I IKi⥯⥯αKi EI+S ⇌αKmEIS →βkcat EI+PEquation EScheme_1 where E, S, and I stand for enzyme, substrate, and 30bpDNA, respectively,K i is the inhibition constant and α and β are dimensionless numbers. The inhibition is said to be partially competitive if ∞ > α > 1 and β = 1 and partially noncompetitive if α = 1 and 0 < β < 1 (18.$$$$$$ ref data missingGoogle Scholar). To decide between these two mechanisms we have measured the kinetic parameters for the hydrolysis of Suc-Ala2-Pro-Phe-pNA by free cat G to getk cat and K m and by30bpDNA-saturated cat G to obtain βk cat and αKm . The following constants were found: k cat, 3.1 ± 0.3 s−1; βk cat, 1.4 ± 0.1 s−1; K m, 2.5 ± 0.2 mm; αKm , 2.8 ± 0.2 mm. Thus, α = 1.1 ± 0.2 and β = 0.45 ± 0.09, which strongly suggests partially noncompetitive inhibition, that is 30bpDNA does not alter the binding of Suc-Ala2-Pro-Phe-pNA to the active site of cat G but hinders its breakdown into products. Szedlacsek et al. (19.Szedlacsek S.E. Ostafe V. Serban M. Vlad M.O. Biochem. J. 1988; 254: 311-312Crossref PubMed Scopus (23) Google Scholar) have derived an equation that allows the estimation of K i from partial tight binding inhibition such as that shown in Fig. 3. This complex equation (not shown) contains five unknown parameters: K i,k cat, K m, α, and β. We have taken the experimentally determined k cat,K m, α, and β terms as known constants of Szedlacsek's equation to calculate K i by nonlinear regression analysis. With the data collected with 24 nm cat G in Fig. 3, K i was 6.7 ± 0.5 nm,whereas with cat G, 300 nm, K i = 11 ± 5 nm. These figures are in excellent agreement with the fluorometrically measured equilibrium dissociation constant.30bpDNA also partially inhibited the activity of cat G on insoluble elastin; the percentage of cat G inhibition was 16.2 ± 4.1. Fig. 4 shows that 30bpDNA sharply depresses the rate constant for the inhibition of cat G by ACT and α1PI. With ACT the maximal decrease was 210-fold, whereas it was 3100-fold in the case of α1PI. The inhibition of cat G by ACT was also tested with variable concentrations of full-length DNA (0.5–10 μg/ml). The rate constant decreased 200-fold as with 30bpDNA (data not shown), indicating that 30bpDNA is a valuable model of full-length DNA. To study the influence of substrate concentration on the inhibition of30bpDNA-bound cat G by ACT and α1PI, we used constant concentrations of enzyme and inhibitor (30 nm cat G + 0.5 μm30bpDNA + 0.5 μm ACT or 10 nm cat G + 0.5 μm30bpDNA + 50 μm α1PI) and variable concentrations of Suc-Ala2-Pro-Phe-pNA (0.8–6 mm). We found that 1/k was linearly related to [S]o, as predicted by Equations 4and 5. As shown in Fig. 5, the rate constant for the inhibition of 30bpDNA-bound cat G varies hyperbolically with the ACT concentration, suggesting two-step inhibition. Indeed, the data could be fit to Equation 5 by nonlinear regression analysis. In contrast, the linear variation of k with the α1PI concentrations indicates one-step inhibition. The kinetic constants are reported in Table I together with those collected previously with free cat G (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar). It can be seen that30bpDNA lowers the second-order rate constant for the inhibition of cat G by ACT and α1PI by a factor of 195 and 3200, respectively.Table IKinetic constants describing the inhibition of neutrophil cat G by ACT and α1PI in the absence and presence of 30bpDNA at pH 7.4 and 25 °CInhibitor30bpDNAInhibition kineticsK i*k 2k 2/K i* or k assms −1m −1 s −1ACTaFrom Duranton et al. (12).−two-step6.2 × 10−82.8 × 10−24.5 × 103ACT+two-step2.6 × 10−66.0 × 10−32.3 × 103α1PIaFrom Duranton et al. (12).−two-step8.1 × 10−75.5 × 10−26.7 × 104α1PI+one-step>1.7 × 10−5bAssuming that in fact the inhibition occurs in two steps with K i*(1 + [S]o/K m) greater than the maximum inhibitor concentration used in the experiment (see also text).>3.6 × 10−4bAssuming that in fact the inhibition occurs in two steps with K i*(1 + [S]o/K m) greater than the maximum inhibitor concentration used in the experiment (see also text).21The errors on the kinetic parameters are less than 15% (experimental constants) or 30% (calculated constants) and are not reported.a From Duranton et al. (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar).b Assuming that in fact the inhibition occurs in two steps with K i*(1 + [S]o/K m) greater than the maximum inhibitor concentration used in the experiment (see also text). Open table in a new tab The errors on the kinetic parameters are less than 15% (experimental constants) or 30% (calculated constants) and are not reported. Two experiments were run to further illustrate the dramatic effect of30bpDNA on the rate inhibition of cat G by α1PI. First, we have incubated constant concentrations of free or polynucleotide-bound cat G with increasing concentrations of α1PI for 1 h, a time largely sufficient to ensure full inhibition of the free enzyme. The inhibitor yielded a linear inhibition curve with free cat G, whereas the 30bpDNA-cat G complex was fully resistant to inhibition (data not shown). Second, we have studied the influence of 30bpDNA on the inhibition of the elastolytic activity of cat G by α1PI. We have found that α1PI fully inhibited the elastolytic activity of cat G in the absence of 30bpDNA, whereas in the presence of the polynucleotide, there was only 28% inhibition. Fig. 6 shows that the progress curves run in the presence of cat G + MPI ± 30bpDNA are biphasic, as predicted for reversible inhibition (Equation 6). The rate constant of inhibition is linearly related to the MPI concentration, indicating one-step inhibition (Equation 8). The kinetic constants k ass, k diss, and K i were found to be 1.5 × 105m−1 s−1, 1.7 × 10−2 s−1, and 1.2 × 10−7m, respectively. MPI also inhibits free cat G in a one-step reaction characterized by k ass = 1.0 × 105m−1 s−1,k diss = 3.5 × 10−3s−1 and K i = 3.5 × 10−8m. Thus, the polynucleotide has virtually no effect on k ass and moderately increasesk diss and K i. Z-Gly-Leu-Phe-CH2Cl is an irreversible cat G inhibitor, whereas chymostatin inhibits the enzyme reversibly (20.Stein R. Strimpler A. Biochemistry. 1987; 26: 2611-2615Crossref PubMed Scopus (40) Google Scholar). The rates of inhibition were measured using 5–10 nmcat G, 0.5 μm30bpDNA, 0.6 mmSuc-Ala2-Pro-Phe-thiobenzylester, and variable concentrations of inhibitor. For both compounds the rate constant of inhibition k was found to be linearly related to the inhibitor concentration, indicating one-step inhibition. Z-Gly-Leu-Phe-CH2Cl inhibited cat G with ak ass of 25 m−1s−1 and 23.9 m−1 s−1in the absence and presence of polynucleotide, respectively. The kinetic parameters for the cat G-chymostatin interaction were found to be: k ass = 2.4 × 104m−1 s−1,k diss = 1.2 × 10−2s−1, in the absence of 30bpDNA andk ass = 1.8 × 104m−1 s−1,k diss = 1.6 × 10−2s−1 in its presence. The polynucleotide does not, therefore, significantly affect the inhibition of cat G by these synthetic inhibitors. Proteinase 3 did not bind to the Sepharose-30bpDNA column even at low ionic strength (50 mm Hepes, pH 7.4). Also, its activity on methoxysuccinyl-lysyl (2-picolinoyl)-Ala-Pro-Val-pNA and on elastin was unaffected by 30bpDNA. On the other hand the polynucleotide did not significantly change its rate of inhibition by α1PI (data not shown). We have explored the possibility that DNA released from neutrophils at sites of inflammation interferes with the inhibition of neutrophil cat G and proteinase 3 by their endogenous inhibitors. A30bpDNA fragment used as a model of DNA was able to tightly bind cat G to form an enzymatically active complex. This complex was almost as active on synthetic substrates and on elastin as free car G and could be easily inhibited by MPI and synthetic cat G inhibitors. In contrast, the 30bpDNA-cat G complex was virtually resistant to inhibition by the two serpins ACT and α1PI. ACT inhibits both free (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar) and 30bpDNA-bound cat G via a two step reaction (see Eq. 3). The polynucleotide decreases both the affinity of the Michaelis-type complex EI* (K i* increases 42-fold) and the rate constant for its conversion into the final complex EI (k 2 decreases ∼15-fold) (Table I). Whereas α1PI inhibits free cat G in two steps (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar), it reacts in one step with the 30bpDNA-cat G complex. The value ofk ass (21 m−1s−1) is, however, several orders of magnitude lower than the maximum rate constant for a bimolecular diffusion-controlled reaction (21.Alberty M.L. Hammes G.G. J. Phys. Chem. 1958; 62: 154-159Crossref Scopus (271) Google Scholar). It may, therefore, be assumed that the reaction involves an intermediate even if the latter is not seen kinetically. This assumption predicts that the largest inhibitor concentration used (50 μm) must be lower than K i* (1 + [S]o/K m) (see Equation 5). Hence, K i* > 17 μm andk 2 > 3.6 × 10−4s−1. Comparison of these limits withK i* and k 2 previously determined with free cat G (12.Duranton J. Adam C. Bieth J .G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (57) Google Scholar) indicates that 30bpDNA increases K i* at least 21-fold and decreasesk 2 at least 153-fold. Thus, the dramatic decrease in the rate of inhibition of cat G by ACT and α1PI is due in both cases to an unfavorable effect of30bpDNA on K i* andk 2. We have previously shown that polynucleotides also depress the rate of neutrophil elastase inhibition by α1PI. For instance, tRNA and polydeoxycytosine decrease the second-order rate constant of inhibition by factors of 3 and 31, respectively (9.Belorgey D. Bieth J.G. Biochemistry. 1998; 37: 16416-16422Crossref PubMed Scopus (27) Google Scholar). We have confirmed these mild effects using 30bpDNA, which depressed the second-order rate constant by a factor of 9 (data not shown). Thus, polynucleotides affect much less the inhibition of elastase by α1PI than that of cat G by α1PI. This is reminiscent of previous findings showing that heparin, another anionic ligand, decreases the rate of inhibition of cat G by α1PI by a factor of 400 (22.Ermolieff J. Boudier C. Laine A. Meyer B. Bieth J.G. J. Biol. Chem. 1994; 269: 29502-29508Abstract Full Text PDF PubMed Google Scholar), whereas it lowers the rate of inhibition of elastase by α1PI by a factor of only 5 (23.Faller B. Cadène M. Bieth J.G. Biochemistry. 1993; 32: 9230-9235Crossref PubMed Scopus (38) Google Scholar). DNA and heparin have thus similar effects on the two proteinase/α1PI systems; they enormously decrease the rate of inhibition of cat G but only marginally affect the rate of inhibition of elastase. This difference may be related to differences in the localization of the arginine residues in cat G and elastase. Arginine residues have the potential to form salt bridges with the anionic groups of heparin and DNA. Unlike elastase (24.Bode W. Wei A.Z. Huber R. Meyer E. Travis J. Neumann S. EMBO J. 1986; 5: 2453-2458Crossref PubMed Scopus (279) Google Scholar), cat G has three arginine residues in the immediate vicinity of its active site (25.Hof P. Mayr I. Huber R. Korzus E. Potempa J. Travis J. Powers J.C. Bode W. EMBO J. 1996; 15: 5481-5491Crossref PubMed Scopus (130) Google Scholar). As a consequence, heparin or 30bpDNA might bind closer to the active site of cat G than to that of elastase thus causing a more steric hindrance to the access of α1PI in cat G than in elastase. Proteinase 3 is not inhibited by ACT or MPI but is rapidly inactivated by α1PI (k ass = 8 × 106m−1 s−1 (7.Rao N. Wehner N. Marshall B. Gray W. Gray B. Hoidal J. J. Biol. Chem. 1991; 266: 9540-9548Abstract Full Text PDF PubMed Google Scholar)). This reaction rate is not altered by 30bpDNA. Thus, the inhibition of the three neutrophil serine proteinases by α1PI is diversely affected by 30bpDNA; cat G almost fully resists inhibition, elastase is inhibited with a moderately reduced rate, and the inhibition of proteinase 3 is not affected at all by the polynucleotide. We believe that our kinetic data have pathological bearing. In chronic bronchitis there is a continuous recruitment of neutrophils in airways. During activation or phagocytosis, neutrophils release part of their cat G, elastase, and proteinase 3 content in airway secretions. In addition, when these short lived cells die in situ they release both their proteinase and DNA content (26.Potter J. Spector S. Matthews L. Lemm J. Am. Rev. Respir. Dis. 1969; 99: 909-916PubMed Google Scholar). We have shown that DNA forms a tight complex with cat G, and so it renders this proteinase virtually resistant to inhibition by the fast acting serpins α1PI and ACT. Thus, DNA promotes cat G-mediated proteolysis of lung matrix proteins in an inhibitory environment. We thank Synergen and Novartis for the gifts of recombinant α1PI and MPI." @default.
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