Title: Interaction between Human Cathepsins K, L, and S and Elastins
Abstract: Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins. Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins. Elastic fibers are the key extracellular matrix component conferring elasticity to tissues such as blood vessels, lungs, and skin. The fibers are composed of a rubber-like network of highly stable and hydrophobic polymers of the protein elastin, associated with peripheral microfibrils (1Rosenbloom J. Lab. Investig. 1984; 51: 605-623PubMed Google Scholar, 2Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar). Proteolytic degradation of elastic fibers leads to loss of tissue elasticity, which is associated with the development of different pathological conditions. A large repertoire of elastinolytic peptidases has been identified in human cells and tissues, including enzymes from four of the five catalytic classes of peptidases. The molecular mechanism underlying the process of elastinolysis has been thoroughly studied for human leukocyte elastase (HLE) 2The abbreviations used are: HLE, human leukocyte elastase; Tg1 domain, thyroglobulin type 1 domain; SSA, specific surface area; DTT, dithiothreitol. 2The abbreviations used are: HLE, human leukocyte elastase; Tg1 domain, thyroglobulin type 1 domain; SSA, specific surface area; DTT, dithiothreitol. (3Baici A. Grassi C. Travis J. Casali L. Luisetti M. Biochemistry of Pulmonary Emphysema. 1992: 81-100Google Scholar, 4Hornebeck W. Schnebli H.P. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 455-458PubMed Google Scholar, 5Robert B. Hornerbeck W. Robert L. Biochimie (Paris). 1974; 56: 239-244Crossref PubMed Scopus (20) Google Scholar, 6Kramps J.A. Morrison H.M. Burnett D. Dijkman J.H. Stockley R.A. Scand. J. Clin. Lab. Investig. 1987; 47: 405-410Crossref PubMed Scopus (8) Google Scholar, 7Hornebeck W. Brechemier D. Jacob M.P. Frances C. Robert L. Adv. Exp. Med. 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Numerous studies have implicated cathepsins K, L, and S, as well as some others, in extensive degradation of elastic fibers, which accompanies the development of pathological conditions of the cardiovascular system. In mice, cathepsin S was found to co-localize with regions of increased elastin breakdown in atherosclerotic plaques (22Rodgers K.J. Watkins D.J. Miller A.L. Chan P.Y. Karanam S. Brissette W.H. Long C.J. Jackson C.L. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 851-856Crossref PubMed Scopus (99) Google Scholar), and reduced atherosclerosis was observed in cathepsin S- and K-deficient mice (23Sukhova G.K. Zhang Y. Pan J.H. Wada Y. Yamamoto T. Naito M. Kodama T. Tsimikas S. Witztum J.L. Lu M.L. Sakara Y. Chin M.T. Libby P. Shi G.P. J. Clin. Investig. 2003; 111: 897-906Crossref PubMed Scopus (318) Google Scholar, 24Lutgens E. Lutgens S.P. Faber B.C. Heeneman S. Gijbels M.M. de Winther M.P. Frederik P. van der Made I. Daugherty A. Sijbers A.M. Fisher A. Long C.J. Saftig P. Black D. Daemen M.J. Cleutjens K.B. Circulation. 2006; 113: 98-107Crossref PubMed Scopus (192) Google Scholar). In rats, up-regulation of cathepsin S and K activity was also observed following carotid artery injury (25Cheng X.W. Kuzuya M. Sasaki T. Arakawa K. Kanda S. Sumi D. Koike T. Maeda K. Tamaya-Mori N. Shi G.P. Saito N. Iguchi A. Am. J. Pathol. 2004; 164: 243-251Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In human atheroma, cathepsins S and K were up-regulated in macrophages as well as smooth muscle cells (26Sukhova G.K. Shi G.P. Simon D.I. Chapman H.A. Libby P. J. Clin. Investig. 1998; 102: 576-583Crossref PubMed Scopus (562) Google Scholar, and increased cathepsin S levels were detected in sera of patients suffering from atherosclerosis and diabetes (27Liu J. Ma L. Yang J. Ren A. Sun Z. Yan G. Sun J. Fu H. Xu W. Hu C. Shi G.P. Atherosclerosis. 2006; 186: 411-419Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Human macrophages in atherosclerotic plaques were shown to degrade elastin extracellularly as well as intracellularly. Cathepsins K and S were attributed major roles in extracellular degradation, whereas cathepsin V was identified as the major peptidase involved in lysosomal digestion (20Yasuda Y. Li Z. Greenbaum D. Bogyo M. Weber E. Broömme D. J. Biol. Chem. 2004; 279: 36761-36770Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Increased levels of cathepsins K, S, and V were also observed in stenotic aortic valves (28Helske S. Syvaranta S. Lindstedt K.A. Lappalainen J. Oorni K. Mayranpaa M.I. Lommi J. Turto H. Werkkala K. Kupari M. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1791-1798Crossref PubMed Scopus (86) Google Scholar), whereas cathepsin S has been connected to elastic lamina fragmentation in hypertensive heart failure (29Cheng X.W. Obata K. Kuzuya M. Izawa H. Nakamura K. Asai E. Nagasaka T. Saka M. Kimata T. Noda A. Nagata K. Jin H. Shi G.P. Iguchi A. Murohara T. Yokota M. Hypertension. 2006; 48: 979-987Crossref PubMed Scopus (84) Google Scholar). Similarly, increased cathepsin L levels were detected in abdominal aortic aneurysm, atherosclerosis, and stenosis (30Liu J. Sukhova G.K. Yang J.T. Sun J.S. Ma L.K. Ren A. Xu W.H. Fu H.X. Dolganov G.M. Hu C.C. Libby P. Shi G.P. Atherosclerosis. 2006; 184: 302-311Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Pathological elastin degradation by cysteine cathepsins has also been implicated in diseases of the pulmonary system. Alveolar macrophages are known to express cathepsins L and S (21Shi G.P. Munger J.S. Meara J.P. Rich D.H. Chapman H.A. J. Biol. Chem. 1992; 267: 7258-7262Abstract Full Text PDF PubMed Google Scholar, 31Reilly Jr., J.J. Mason R.W. Chen P. Joseph L.J. Sukhatme V.P. Yee R. Chapman Jr., H.A. Biochem. J. 1989; 257: 493-498Crossref PubMed Scopus (58) Google Scholar), whereas cathepsin K was shown to be expressed by lung epithelial cells (32Buöhling F. Gerber A. Hackel C. Kruger S. Kohnlein T. Broömme D. Reinhold D. Ansorge S. Welte T. Am. J. Respir. Cell Mol. Biol. 1999; 20: 612-619Crossref PubMed Scopus (83) Google Scholar). Increased cathepsin L activity was observed in bronchoalveolar lavage fluid of smokers (33Takahashi H. Ishidoh K. Muno D. Ohwada A. Nukiwa T. Kominami E. Kira S. Am. Rev. Respir. Dis. 1993; 147: 1562-1568Crossref PubMed Google Scholar), and increased levels of cathepsins B, H, K, L, and S were observed in bronchopulmonary dysplasia (34Altiok O. Yasumatsu R. Bingol-Karakoc G. Riese R.J. Stahlman M.T. Dwyer W. Pierce R.A. Broömme D. Weber E. Cataltepe S. Am. J. Respir. Crit. Care Med. 2006; 173: 318-326Crossref PubMed Scopus (61) Google Scholar). In vivo, proteolytic activity of cysteine cathepsins is regulated by endogenous inhibitors, including the general cysteine cathepsin inhibitors cystatins (35Barrett A.J. Biomed. Biochim. Acta. 1986; 45: 1363-1374PubMed Google Scholar), the more specific thyropins (36Lenarčič B. Bevec T. Biol. Chem. 1998; 379: 105-111PubMed Google Scholar), and several others. The importance of the peptidase/inhibitor balance has been well investigated in tumor progression (reviewed in Ref. 37Skrzydlewska E. Sulkowska M. Koda M. Sulkowski S. World J. Gastroenterol. 2005; 11: 1251-1266Crossref PubMed Scopus (133) Google Scholar). Reduced cystatin C levels have also been observed in atherosclerosis and aortic aneurysm in human and mouse (38Shi G.P. Sukhova G.K. Grubb A. Ducharme A. Rhode L.H. Lee R.T. Ridker P.M. Libby P. Chapman H.A. J. Clin. Investig. 1999; 104: 1191-1197Crossref PubMed Scopus (412) Google Scholar, 39Sukhova G.K. Wang B. Libby P. Pan J.H. Zhang Y. Grubb A. Fang K. Chapman H.A. Shi G.P. Circ. Res. 2005; 96: 368-375Crossref PubMed Scopus (135) Google Scholar, 40Bengtsson E. To F. Hakansson K. Grubb A. Branen L. Nilsson J. Jovinge S. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2151-2156Crossref PubMed Scopus (98) Google Scholar), and imbalance between cysteine cathepsins and their inhibitors was also reported in bronchopulmonary dysplasia (34Altiok O. Yasumatsu R. Bingol-Karakoc G. Riese R.J. Stahlman M.T. Dwyer W. Pierce R.A. Broömme D. Weber E. Cataltepe S. Am. J. Respir. Crit. Care Med. 2006; 173: 318-326Crossref PubMed Scopus (61) Google Scholar). In existing reports on the elastinolytic activity of cysteine cathepsins, the mechanism utilized by these peptidases to degrade elastin has not yet been investigated. In this work, we have studied the interaction between elastin and the human cathepsins K, L, and S, which have been most frequently implicated in extracellular elastinolysis. Because the structural organization of elastic fibers differs between tissues, we used bovine elastins from three different sources as follows: aorta, lung, and neck ligament. We investigated the mode of cathepsin binding to elastin, the overall elastinolytic activity, and the effect of macromolecular inhibitors from the cystatin and thyropin families on the elastinolytic activity of cysteine cathepsins. Elastins, Enzymes, and Inhibitors—All elastin types were purchased from Elastin Products Co., Inc. The elastins used in this study were all of bovine origin as follows: neck ligament elastin (number E70; particle size, 100–400 mesh), aortic elastin (number SB87; particle size, pass 100 mesh), lung elastin (number SB77; particle size, pass 100 mesh), and ETNA-elastin (number E60; prepared from neck ligament, salt-free, water-soluble). Recombinant human cathepsin K (EC 3.4.22.38), cathepsin L (EC 3.4.22.15), and cathepsin S (EC 3.4.22.27) were produced in Escherichia coli according to published procedures (41Dolinar M. Maganja D.B. Turk V. Biol. Chem. Hoppe-Seyler. 1995; 376: 385-388Crossref PubMed Scopus (37) Google Scholar, 42Kopitar G. Dolinar M. ŝtrukelj B. Pungerčar J. Turk V. Eur. J. Biochem. 1996; 236: 558-562Crossref PubMed Scopus (41) Google Scholar, 43D'Alessio K.J. McQueney M.S. Brun K.A. Orsini M.J. Debouck C.M. Protein Expression Purif. 1999; 15: 213-220Crossref PubMed Scopus (30) Google Scholar). Active concentrations of all enzymes were determined by active site titrations with the irreversible inhibitor E-64 (Bachem, Switzerland). Recombinant human stefin A was produced according to Ref. 44Pol E. Olsson S.L. Estrada S. Prasthofer T.W. Bjork I. Biochem. J. 1995; 311: 275-282Crossref PubMed Scopus (37) Google Scholar, and the cystatin-like domain 3 of human kininogen (kininogen domain 3) was isolated as described previously (45Lenarčič B. Kraŝovec M. Ritonja A. Olafsson I. Turk V. FEBS Lett. 1991; 280: 211-215Crossref PubMed Scopus (85) Google Scholar) and recombinant thyroglobulin type 1 (Tg1) domain of human testican-1 as described previously (46Meh P. Pavŝič M. Turk V. Baici A. Lenarčič B. Biol. Chem. 2005; 386: 75-83Crossref PubMed Scopus (26) Google Scholar). The recombinant human p41 fragment of the major histocompatibility complex class II-associated invariant chain, recombinant Tg1 domain of human nidogen-1, and recombinant Tg1 domain 1 of human nidogen-2 were produced in-house. In brief, cDNA sequences coding for individual Tg1 domains were amplified by PCR and cloned into the pET-32b(+) expression vector (Novagen, Germany) using the NcoI and XhoI restriction sites. Soluble thioredoxin fusion proteins were then expressed in E. coli strain BL21 (DE3) pLysS (Novagen, Germany) at 37 °C. Fusion proteins were purified from cell lysates by immobilized nickel ion-affinity chromatography. The Tg1 domains were liberated from the fusion proteins by enterokinase cleavage and purified by ion-exchange chromatography. Samples were over 95% pure as visualized by SDS-PAGE. Active p41 fragment concentration was determined by active site titration of cathepsin L, and the concentrations of the nidogen Tg1 domains were determined spectrophotometrically at 280 nm. Elastin Surface Area Determination and Scanning Electron Microscopy—The specific surface area (SSA) of solid and powdered elastins was measured by nitrogen adsorption at –196 °C using the Brunauer et al. theory (47Brunauer S. Emmett P.H. Teller E. J. Am. Chem. Soc. 1938; 60: 309-319Crossref Scopus (21238) Google Scholar) (BET-method, Tristar, Micrometrics Inc, Gemini, The Netherlands). An eight point isotherm, 0.02 < p/p0 < 0.2), was used after degassing the samples at room temperature for 1 h. The morphology of the elastin samples was analyzed by scanning electron microscopy using a LEO 1530 Gemini (Oberkochen, Germany). Kinetic Measurements—Binding of cathepsins to insoluble elastin was studied using an approach originally developed for describing the interaction of HLE with macromolecular substrates (9Baici A. Biochim. Biophys. Acta. 1990; 1040: 355-364Crossref PubMed Scopus (20) Google Scholar). With this method the hydrolysis of a low molecular mass fluorogenic substrate (called the reporter substrate) is measured in the presence of various amounts of a macromolecular substrate. In the assay, the enzyme is partitioned between the macromolecular substrate and the reporter substrate, and the perturbation caused by the macromolecular substrate to the kinetics of reporter substrate hydrolysis can be formally treated as that of an inhibitor. Kinetic measurements were performed in a C-61 fluorimeter (Photon Technology International) at 25 °C. Assays with cathepsins K and L were performed in 50 mm sodium phosphate buffer, pH 6.50, containing 2.5 mm EDTA and using the fluorogenic substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Bachem, Switzerland). In cathepsin K assays, the final concentration of the reporter substrate was 5 μm (Km = 7.5 μm), and in cathepsin L assays the concentration was 10 μm (Km = 0.93 μm). Assays with cathepsin S were performed in 50 mm sodium phosphate buffer, pH 6.50, containing 2.5 mm EDTA and 0.1% PEG-6000, with benzyloxycarbonyl-Phe-Val-Arg-7-amino-4-methylcoumarin as the substrate (Bachem, Switzerland) at a final concentration of 10 μm (Km = 12.5 μm). Active enzyme concentrations in the assays were 60 pm for cathepsin K, 200 pm for cathepsin L, and 2 nm for cathepsin S. Variable amounts of elastin powder (ranging from 0.2 to 15 mg) were introduced into disposable 10 × 10-mm acryl cuvettes. 2 ml of the appropriate assay buffer were added to the powder, and the cuvette was then hermetically sealed and incubated overnight at room temperature. Immediately prior to the experiment, the mixture was supplemented with the appropriate substrate and DTT (final concentration 2.5 mm). Reaction was started by addition of the enzyme, and the progress was monitored continuously for 3 min at λex = 383 nm and λem = 455 nm. To minimize light scattering, narrow excitation and emission bandwidths of 1 nm were used. During the assay the reaction mixture was continuously stirred using a built-in magnetic stirrer and a Teflon-coated stirring rod to avoid sedimentation of the insoluble material. Assays with soluble ETNA-elastin were performed under the conditions described above. A stock elastin solution (10 mg/ml) was prepared by dissolving ETNA-elastin in the appropriate assay buffer and diluted immediately prior to the experiment. Kinetic Models for Cathepsin Binding to Elastins—To describe the binding of cysteine cathepsins to the surface of insoluble elastin, we propose three models of interaction, which are shown in Fig. 1. The three models and their corresponding equations are described with terminology and dimensions typical of interfacial enzyme catalysis, with the asterisk denoting processes occurring at the surface of insoluble elastin (48Berg O.G. Jain M.K. Interfacial Enzyme Kinetics. 2002; (John Wiley & Sons Ltd., Chichester, UK): 47-75Google Scholar). A*M and K* represents the elastin surface area per unit volume and the interfacial dissociation constant, respectively, both with dimensions m2·liter–1; [S] is the molar concentration of the reporter substrate, and Km is its Michaelis constant. The models consist of two parts as follows: the upper parts represent the Michaelis-Menten path of reporter substrate hydrolysis, and the lower parts represent the adsorption of the enzyme to the matrix, followed by a catalytic step, which is shown in Fig. 1 as dashed boxes in each model. This part is not visible in the assays, because the elastin cleavage products (Q) are deprived of a spectroscopically measurable signal. Therefore, the macromolecular substrate can be formally regarded as a competitive inhibitor, and the measured "inhibition constant" of the macromolecular substrate (M) actually yields an analogous of the Michaelis constant. However, by using low enzyme concentrations and short reaction times, we can safely assume that the perturbation on the formation of the fluorescent product (P) caused by M yields a good estimate of the dissociation constant K*. In Model 1 the behavior of the macromolecular substrate is analogous to that of a linear competitive inhibitor and corresponds to enzyme adsorption to the matrix in a nonproductive manner followed by isomerization to a catalytically productive complex. The treatment of Models 2 and 3 is analogous to that of a competitive inhibitor, two molecules of which bind the enzyme in an ordered sequence. Model 3 differs from Model 2 by a dimensionless coefficient a that multiplies K* and describes a change of the dissociation constant for the vacant site following the adsorption step, thus taking into account the cooperativity of the binding process. In Fig. 1, Model 1 is described by Equation 1, which is equivalent to the equation for linear competitive inhibition; Model 2 is described by Equation 2, and for Model 3 Equation 3 applies. The interaction of cathepsins with soluble ETNA-elastin was analyzed using the same models, except that elastin surface area per unit volume (A*M) was replaced by mass concentration [M] in the corresponding equations, and the interface equilibrium dissociation constant K*, used to describe the adsorption of cathepsins to the surface of insoluble elastin, was replaced by the equilibrium dissociation constant in solution K (given in g·liter–1). Discrimination between the models was performed by fitting the corresponding Equations 1–3 in Fig. 1 to experimental data using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego. First, Equation 1 was fitted to the data, and the runs test was performed to check any systematic deviation of the fitted curve from the data. If there was no systematic deviation, a discrimination test was performed by analysis of variance of the difference between the sum of squares of Models 1 and 2 (extra sum-of-squares test). An F ratio and a p value were calculated as follows: for a p value less than 0.05, the more complicated model (Model 2) was chosen as appropriate. If fitting of Equation 1 did not pass the runs test, discrimination was performed with the extra-sum-of squares test applied to Model 2 and Model 3. An additional tool for interpreting the kinetics of cathepsin binding to elastin was the 90:10 ratio (A*M,0.9/A*M,0.1), i.e. the ratio of elastin surface areas necessary to reduce enzyme activity by 90 and 10%. For Model 1, the 90:10 ratio is always 81 (Equation 4), a characteristic of linear competitive inhibitors (49Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. 1975; (Wiley Interscience, New York): 106Google Scholar). For Model 2, the 90:10 ratio depends on the [S]/Km ratio (Equation 5), and for Model 3 it depends on the values of [S]/Km and a (see Equation 6). Elastinolytic Assays—The elastinolytic activities of cathepsins K, L, and S were measured using a modified procedure of Schwabe (50Schwabe C. Anal. Biochem. 1973; 53: 484-490Crossref PubMed Scopus (63) Google Scholar). In brief, reaction mixtures containing 1.0 mg of insoluble elastin in a total volume of 200 μl of the appropriate assay buffer supplemented with 2.5 mm DTT (see under "Kinetic Measurements") were prepared. Reactions were started by addition of the enzyme and incubated in an Eppendorf Thermomixer Compact at 37 °C with shaking (1200 rpm) to avoid sedimentation. Enzyme concentrations in the assays were 285 nm cathepsin K, 100 nm cathepsin L, and 100 nm cathepsin S. The reaction mixtures were incubated for 10–120 min, and the reactions then stopped by addition of trichloroacetic acid to a final concentration of 5% (w/v). The samples were centrifuged for 15 min at 14,000 × g, and 100 μl of clear supernatant were added to 3.0 ml of 0.2 m sodium borate buffer, pH 8.5, and then combined with 1.0 ml of a fluorescamine solution (15 mg/100 ml in acetone) under vigorous stirring. The fluorescence of the labeled peptides was measured at λex = 390 nm and λem = 480 nm. Appropriate blanks were run to take into account the fluorescence developed by elastin and enzyme alone. The measured fluorescence was compared with a standard curve, which was produced using glycine solutions of known concentrations. The rate of peptide release from elastin was determined as the slope of the curve obtained from multiple samples incubated for increasing periods of time and was then normalized to 1 pmol of enzyme. Adsorption of Cathepsins on Elastin—The adsorption of cathepsins K, L, and S on elastin was studied by a modified procedure of Ying and Simon (12Ying Q.L. Simon S.R. Am. J. Respir. Cell Mol. Biol. 2002; 26: 356-361Crossref PubMed Scopus (21) Google Scholar). The partitioning of the enzyme between the insoluble phase (elastin) and soluble phase (aqueous buffer) was studied in the presence of excess amounts of elastin to allow for concentration-independent adsorption of the enzyme. In brief, 100 μl of an elastin suspension (10 mg/ml) in 50 mm sodium phosphate buffer, pH 6.50, containing 2.5 mm EDTA and 2.5 mm DTT, was incubated with each cathepsin (concentrations ranging from 0.1 to 1 μm) for 10 min at 25 °C and with shaking to avoid sedimentation. The samples were then centrifuged for 3 min at 14,000 × g, and the enzymatic activity of the supernatant was measured fluorimetrically as described under "Kinetic Measurements." In parallel, control samples without elastin were run and were used to calculate the percentage of enzyme adsorbed to the elastin surface. Inhibition of Elastinolysis—The proteinaceous inhibitors used in this study were recombinant stefin A, kininogen domain 3, Tg1 domain of testican-1, the p41 fragment, Tg1 domain of nidogen-1, and Tg1 domain 1 of nidogen-2. Experiments were performed as described under "Elastinolytic Assays," except that inhibitors were added to the mixture either prior to addition of the enzyme or following a 30-min preincubation of the enzyme with elastin. All samples were incubated for a total of 60 min, and released peptides then detected by reacting them with fluorescamine as described under "Elastinolytic Assays." Appropriate blanks were run to account for fluorescence of elastin, enzyme, and inhibitor alone. SSA and Microscopic Analysis of Elastin Particles—Bovine elastins from three different anatomical regions were analyzed by scanning electron microscopy to verify their morphological features as a support to the interpretation of our kinetic measurements (Fig. 2). Neck ligament elastin appeared predominantly as extended patches, or splinters thereof, with a rough surface, which was spotted by small fragments plausibly generated during extraction from the tissue and sample preparation (Fig. 2C). These small fragments are likely to substantially contribute to the SSA of neck ligament elastin (2.3 m2/g). In contrast, lung elastin showed a distinct three-dimensional network composed of bundles of fibers (Fig