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• W1964855331 abstract "Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K m values in the nanomolar range and relative low k catvalues (1.9 × 10−3 and 4.1 × 10−4s−1, for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1. Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K m values in the nanomolar range and relative low k catvalues (1.9 × 10−3 and 4.1 × 10−4s−1, for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1. matrix metalloproteinase tissue inhibitor of metalloproteinase extracellular matrix polyacrylamide gel electrophoresis surface plasmon resonance phosphate-buffered saline pro-MMP-9 monomer pro-MMP-9 dimer monoclonal antibody polyclonal antibody Matrix metalloproteinase-9 (MMP-9),1 also known as gelatinase B, is a member of the MMP family of zinc-dependent endopeptidases known for their ability to degrade many extracellular matrix (ECM) components (1.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar, 2.Vu T.H. Werb Z. Parks W.C. Mecham R.P. Gelatinase B: Structure, Regulation and Function Matrix Metalloproteinases. Academic Press, San Diego1998: 115-148Google Scholar). MMP-9 is secreted in a latent form (pro-MMP-9) by a variety of normal and transformed cells and has been implicated in the pathogenesis of several human diseases including arthritis (3.Ahrens D. Koch A.E. Pope R.M. Stein-Picarella M. Niedbala M.J. Arthritis Rheum. 1996; 39: 1576-1587Crossref PubMed Scopus (262) Google Scholar), cardiovascular disease (4.Li Y.Y. Feldman A.M. Sun Y. McTiernan C.F. Circulation. 1998; 98: 1728-1734Crossref PubMed Scopus (328) Google Scholar, 5.Tamarina N.A. McMillan W.D. Shively V.P. Pearce W.H. Surgery. 1997; 122: 264-272Abstract Full Text PDF PubMed Scopus (263) Google Scholar), and cancer metastasis (6.Himelstein B.P. Canete-Soler R. Bernhard E.J. Dilks D.W. Muschel R.J. Invasion Metastasis. 1994; 14: 246-258PubMed Google Scholar, 7.Sehgal G. Hua J. Bernhard E.J. Sehgal I. Thompson T.C. Muschel R.J. Am. J. Pathol. 1998; 152: 591-596PubMed Google Scholar). MMP-9 has also been suggested to play a role in the degradation of ECM during inflammation (8.Ohno I. Ohtani H. Nitta Y. Suzuki J. Hoshi H. Honma M. Isoyama S. Tanno Y. Tamura G. Yamauchi K. Nagura H. Shirato K. Am. J. Respir. Cell. Mol. Biol. 1997; 16: 212-219Crossref PubMed Scopus (212) Google Scholar), wound healing (9.Agren M.S. Jorgensen L.N. Andersen M. Viljanto J. Gottrup F. Br. J. Surg. 1998; 85: 68-71Crossref PubMed Scopus (64) Google Scholar, 10.Moses M.A. Marikovsky M. Harper J.W. Vogt P. Eriksson E. Klagsbrun M. Langer R. J. Cell. Biochem. 1996; 60: 379-386Crossref PubMed Scopus (110) Google Scholar), trophoblast implantation (11.Canete-Soler R. Gui Y.H. Linask K.K. Muschel R.J. Dev. Dyn. 1995; 204: 30-40Crossref PubMed Scopus (54) Google Scholar), and angiogenesis (12.Vu T.H. Shipley J.M. Bergers G. Berger J.E. Helms J.A. Hanahan D. Shapiro S.D. Senior R.M. Werb Z. Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1474) Google Scholar). Structurally, pro-MMP-9 is closely related to pro-MMP-2 (gelatinase A) with both enzymes containing a fibronectin-like type II module (gelatin-binding domain) inserted into the catalytic domain that is thought to facilitate interaction of the enzymes with collagen molecules (13.Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (695) Google Scholar, 14.Murphy G. Crabbe T. Methods Enzymol. 1995; 248: 470-484Crossref PubMed Scopus (174) Google Scholar). The zymogenic forms of both enzymes interact, via their C-terminal domain (hemopexin-like domain), with tissue inhibitors of metalloproteinases (TIMPs), a family of specific endogenous MMP inhibitors (15.Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar, 16.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (241) Google Scholar). Pro-MMP-9 binds to TIMP-1 (1.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar), whereas pro-MMP-2 binds to TIMP-2 (17.Goldberg G.I. Marmer B.L. Grant G.A. Eisen A.Z. Wilhelm S. He C.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8207-8211Crossref PubMed Scopus (542) Google Scholar) and to TIMP-4 (18.Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). After activation, any TIMP molecule efficiently inhibits the enzymatic activity by binding to the catalytic domain of the MMP (16.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (241) Google Scholar). Despite the similarities that exist between pro-MMP-9 and pro-MMP-2, the former is unique in several aspects including its gene regulation, structure, and function (2.Vu T.H. Werb Z. Parks W.C. Mecham R.P. Gelatinase B: Structure, Regulation and Function Matrix Metalloproteinases. Academic Press, San Diego1998: 115-148Google Scholar, 14.Murphy G. Crabbe T. Methods Enzymol. 1995; 248: 470-484Crossref PubMed Scopus (174) Google Scholar). Pro-MMP-9 is glycosylated and contains an additional 54-amino acid proline-rich insertion of unknown function between the catalytic and the hemopexin-like domains (1.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). In addition, pro-MMP-9, in contrast to pro-MMP-2, exists in two major forms: a monomeric (∼92 kDa) and a disulfide-bonded homodimeric (∼220 kDa) form (1.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). Both the monomeric and dimeric forms of pro-MMP-9 forms have been identified in a variety of pro-MMP-9-producing cells including normal (19.Kjeldsen L. Johnsen A.H. Sengelov H. Borregaard N. J. Biol. Chem. 1993; 268: 10425-10432Abstract Full Text PDF PubMed Google Scholar, 20.Triebel S. Blaser J. Reinke H. Tschesche H. FEBS Lett. 1992; 314: 386-388Crossref PubMed Scopus (212) Google Scholar, 21.Toth M. Gervasi D.C. Fridman R. Cancer Res. 1997; 57: 3159-3167PubMed Google Scholar) and tumor cells (1.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar, 22.Moll U.M. Youngleib G.L. Rosinski K.B. Quigley J.P. Cancer Res. 1990; 50: 6162-6170PubMed Google Scholar) and in various biological fluids (23.Vartio T. Baumann M. FEBS Lett. 1989; 255: 285-289Crossref PubMed Scopus (79) Google Scholar, 24.Mautino G. Oliver N. Chanez P. Bousquet J. Capony F. Am. J. Respir. Cell. Mol. Biol. 1997; 17: 583-591Crossref PubMed Scopus (204) Google Scholar) and tissues (25.Upadhya A.G. Harvey R.P. Howard T.K. Lowell J.A. Shenoy S. Strasberg S.M. Hepatology. 1997; 26: 922-928Crossref PubMed Scopus (89) Google Scholar, 26.Gonzalez-Avila G. Iturria C. Vadillo-Ortega F. Ovalle C. Montano M. Pathobiology. 1998; 66: 196-204Crossref PubMed Scopus (42) Google Scholar), indicating that they are physiological forms of the enzyme. In addition, a 125–130-kDa form of pro-MMP-9 present in neutrophil granules has been reported to be a complex of the enzyme with lipocalin (NGAL) (19.Kjeldsen L. Johnsen A.H. Sengelov H. Borregaard N. J. Biol. Chem. 1993; 268: 10425-10432Abstract Full Text PDF PubMed Google Scholar, 20.Triebel S. Blaser J. Reinke H. Tschesche H. FEBS Lett. 1992; 314: 386-388Crossref PubMed Scopus (212) Google Scholar). Studies examining the activation, catalytic activity, and interactions with TIMPs of pro-MMP-9 and MMP-9 have focused mainly on the monomeric form of the enzyme. Thus, little is known about the biochemical properties of the homodimeric form. A previous study examined the structural requirements for the formation of the pro-MMP-9 homodimer and its interaction with TIMP-1 (27.Goldberg G.I. Strongin A. Collier I.E. Genrich L.T. Marmer B.L. J. Biol. Chem. 1992; 267: 4583-4591Abstract Full Text PDF PubMed Google Scholar). However, the kinetics of activation, the catalytic efficiencies, and the inhibition by TIMPs of the monomeric and dimeric forms remained unknown. Here, we report the first comprehensive study aimed at characterizing the biochemical properties of both the latent and active pure monomeric and dimeric forms. Buffer C (50 mm HEPES (pH 7.5), 150 mm NaCl, 5 mm CaCl2, and 0.02% Brij-35); buffer B (10 mm sodium acetate (pH 4.5)); buffer W (7.8 mm NaH2PO4, 8 mmNa2HPO4 (pH 7.2), 137 mm NaCl, 0.1 mm CaCl2, 3 mm KCl, 1.5 mm KH2PO4, and 0.02% Tween 20); buffer R (50 mm HEPES (pH 7.5), 150 mm NaCl, 5 mm CaCl2, 0.01% Brij-35, and 1% (v/v) Me2SO); buffer D (50 mm Tris (pH 7.4), 150 mm NaCl, 5 mm CaCl2, and 0.02% Brij-35) and lysis buffer (25 mm Tris-HCl (pH 7.5), 1% Nonidet P-40, 100 mm NaCl, 5 mm EDTA, 20 mm N-ethylmaleimide, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 2 mm benzamidine, and 1 mm phenylmethylsulfonyl fluoride). Human recombinant pro-MMP-9, TIMP-1, and TIMP-2 were produced in mammalian cells using a recombinant vaccinia virus mammalian cell expression system, as described previously (28.Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar). Pro-MMP-9 was purified to homogeneity from the media of infected HeLa cells by gelatin-agarose chromatography, as described previously (29.Fridman R. Toth M. Pena D. Mobashery S. Cancer Res. 1995; 55: 2548-2555PubMed Google Scholar). The concentration of pro-MMP-9 was determined using the molar extinction coefficient of 114,360 m−1 cm−1 (14.Murphy G. Crabbe T. Methods Enzymol. 1995; 248: 470-484Crossref PubMed Scopus (174) Google Scholar). Recombinant TIMP-1 and TIMP-2 were purified as described previously (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Protein concentrations of TIMP-1 and TIMP-2 were determined using their molar extinction coefficients of 26,500 and 39,600m−1 cm−1, respectively (16.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (241) Google Scholar). A sample (7600 pmol) of purified pro-MMP-9 diluted in buffer C was layered onto four polyallomer tubes containing a preformed 20–35% glycerol gradient prepared in buffer C. The tubes were then centrifuged (63 h, 4 °C) in a SW41 rotor at 37,000 rpm, after which nine fractions (∼200 μl each) were collected and assayed for the presence of monomeric or dimeric forms by gelatin-zymography. Fractions containing homogeneous monomeric (pro-MMP-9M) or dimeric (pro-MMP-9D) forms were pooled, and their protein concentrations were determined from the molar extinction coefficients: for pro-MMP-9M, 103,645 m−1cm−1; and for pro-MMP-9D, 198,609m−1 cm−1 (31.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar). TIMP-1 and TIMP-2 were iodinated with carrier free Na125I (100 mCi/ml, Amersham Pharmacia Biotech) using IODOGEN (Pierce) as described previously (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The specific activities of 125I-TIMP-1 and125I-TIMP-2 were calculated to be 0.035 and 0.045 μCi/pmol, respectively. Ten pmol of purified pro-MMP-9M or pro-MMP-9D were each incubated (1 h, 22 °C) with either 125I-TIMP-1 or125I-TIMP-2 (20 pmol with pro-MMP-9M and 30 pmol with pro-MMP-9D) in a final volume of 0.25 ml. The mixtures were then subjected to gel filtration using a Superose-12 column pre-equilibrated with buffer C. As control,125I-TIMP-1 or 125I-TIMP-2 (30 pmol) were chromatographed alone under the same conditions. Fractions (350 μl) were collected and analyzed for radioactivity in a γ counter (Packard model 5650). The amount (picomoles) of 125I TIMP-1 or125I-TIMP-2 bound to the pro-MMP-9 forms was determined from the specific activity. SDS-PAGE was performed according to Laemmli (32.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Proteins were visualized by staining overnight with a 0.25% solution of Coomassie Brilliant Blue R-250 in 45% methanol and 10% acetic acid, and destained in a solution of 20% methanol and 10% acetic acid. Gelatin zymography was performed as described (33.Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar). A fresh tissue biopsy (∼50 mg) (kindly provided by Dr. D. Visscher, Department of Pathology, Harper Hospital, Detroit, MI) of a breast carcinoma was minced into small pieces and resuspended in 500 μl of cold lysis buffer. The pieces were homogenized on ice with a pestle (Kontes, Vineland, NJ) in a microcentrifuge tube, followed by a centrifugation (14,000 rpm) of the homogenate for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined by the BCA protein assay (Pierce). The protein concentration was adjusted to 1 μg/μl of 1× sample buffer, and the sample was then subjected to gelatin zymography as described above and to immunoblot analysis, as described (21.Toth M. Gervasi D.C. Fridman R. Cancer Res. 1997; 57: 3159-3167PubMed Google Scholar), using an anti-MMP-9 rabbit polyclonal antibody (pAb 109) raised against a synthetic peptide (APRQRQSTLVLTPGDLRT) from the prodomain of human pro-MMP-9 (a generous gift from Dr. Stetler-Stevenson, NCI, National Institutes of Health, Bethesda, MD). Monkey kidney BS-C-1 cells (80% confluent) in 60-mm dishes were co-infected with 3 plaque-forming units/cell of vTF7–3 vaccinia virus encoding for T7 RNA polymerase and with 3 plaque-forming units/cell of a recombinant vaccinia virus containing the full-length cDNA of human pro-MMP-9 (vT7-GELB) as described (28.Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar). Four hours after infection, the medium was aspirated and the cell monolayer was gently washed with warm PBS. The cells were incubated (30 min) with 1.5 ml/dish starving medium (Dulbecco's modified Eagle's medium without methionine supplemented with 25 mm Hepes and 0.5% fetal bovine serum). The cells were then pulsed with 500 μCi/ml [35S]methionine in starvation medium (1.5 ml/dish) for 15 min at 37 °C. After the pulse, the dishes were placed on ice, the medium was aspirated and the cells were washed twice with PBS before the addition of 1 ml/dish chase medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 4.8 mm methionine). At the end of the chase periods (0–240 min at 37 °C), the medium was collected; the cells were washed with cold PBS and lysed with 1 ml/dish lysis buffer. The lysates were clarified by a brief centrifugation and were pre-absorbed on protein G-Sepharose beads. The lysates and the media were subjected to immunoprecipitation with either a mAb to pro-MMP-9 (CA-209) or mouse IgG and protein G-Sepharose beads, as described (21.Toth M. Gervasi D.C. Fridman R. Cancer Res. 1997; 57: 3159-3167PubMed Google Scholar). The immunoprecipitates were mixed with Laemmli sample buffer, with or without β-mercaptoethanol, and resolved by 8–16% SDS-PAGE followed by autoradiography. To obtain the active monomer and dimer, purified pro-MMP-9M (100 pmol) or pro-MMP-9D(60 pmol) in buffer C were incubated (2 h at 37 °C) with 25 pmol of heat-activated recombinant human stromelysin 1 (MMP-3, a generous gift from Dr. Paul Cannon, Center for Bone and Joint Research, Palo Alto, CA). The activated monomer (MMP-9M) and dimer (MMP-9D) were then subjected to gelatin-agarose chromatography to remove stromelysin 1, as described (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Fractions containing MMP-9M or MMP-9D were detected by gelatin zymography and pooled. Enzyme concentrations were determined by titration with TIMP-1 and from their native molar extinction coefficients of 99,817 and 191,349 m−1cm−1, respectively, as determined by the method of Gill and von Hippel (31.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar). The activities of the purified MMP-9Mand MMP-9D were assayed using the fluorescence quenched substrate MOCAcPLGLA2pr(Dnp)-AR-NH2 (Peptide Institute, Inc. Japan), as described (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 34.Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (669) Google Scholar). Each assay was carried out at 25 °C in 2 ml (final volume) of buffer R containing enzyme and and/or inhibitor at the indicated concentrations. The substrate concentration was varied from 0.05 to 8.0 μm. The enzyme concentrations were 0.2 and 0.1 nm for MMP-9Mand MMP-9D, respectively. Substrate hydrolysis was monitored using a Photon Technology International (PTI) fluorescence spectrophotometer with excitation and emission wavelengths set at 328 and 393 nm, respectively, controlled by a Pentium™ computer using the RatioMaster™ hardware and FeliX™ software provided by PTI. The excitation and emission band passes were 1 and 3 nm, respectively. Fluorescent measurements were taken with a 4-s integration time. Three initial rate determinations were made for each substrate concentration. The K m and V max values were determined by non-linear regression analyses using GraphPad Prism™ and examined by double-reciprocal analysis by linear regression using LINEST (Microsoft Excel™ version 5.0). Increasing concentrations (0.07–0.3 nm) of active site-titrated (with TIMP-1) MMP-9M and MMP-9D were incubated with 1 μm fluorescein-labeled DQ™ gelatin (Molecular Probes, Eugene, OR) in buffer D, in a total volume of 2 ml. Substrate hydrolysis was monitored over a 1-h period at 25 °C using a PTI spectrofluorometer at excitation and emission wavelengths of 495 and 515 nm, respectively. Excitation and emission band passes were 1 and 3 nm, respectively. Background fluorescence due to DQ™ gelatin was measured with substrate in the absence of enzymes and was subtracted from each trace. A fluorescein (Molecular Probes) standard curve was used to correlate the fluorescence increase with the amount of released fluorescein. Human recombinant pro-stromelysin 1 was heat activated at 55 °C for 1 h. The amount of catalytically competent stromelysin 1 was determined by active-site titration with human recombinant TIMP-1. Stromelysin 1 activity was measured with 5 μm fluorogenic peptide substrate MOCAcRPKPVE-Nva-WRK(Dnp)-NH2 (35.Nagase H. Fields C.G. Fields G.B. J. Biol. Chem. 1994; 269: 20952-20957Abstract Full Text PDF PubMed Google Scholar) (Peptides International, Louisville, KY) in buffer R, at excitation and emission wavelengths of 325 and 393 nm, respectively. Activation of pro-MMP-9M or pro-MMP-9D was monitored in reaction mixtures containing 2–120 nm of either substrate and 0.5 nm stromelysin 1 in 70 μl of buffer D at 37 °C. At varying times, aliquots (20 μl) of the reaction mixture were added to acrylic cuvettes containing 2 ml of 7 μmMOCAcPLGLA2pr(Dnp)-AR-NH2 in buffer R at 25 °C. Less than 10% of hydrolysis of the fluorogenic substrate was monitored, as described by Knight (36.Knight C.G. Methods Enzymol. 1995; 248: 18-34Crossref PubMed Scopus (96) Google Scholar). Hydrolysis of this peptide by stromelysin 1 at the concentrations used (0.5–9 nm) was insignificant when compared with the hydrolysis by MMP-9. The MMP-9 (monomer or dimer) concentrations were calculated using the Michaelis-Menten equation and the k cat andK m values for the reaction of the enzyme (monomer and dimer) with the fluorogenic substrate, as described above. Initial velocities of pro-MMP-9M or pro-MMP-9Dactivation were determined from the linear increase in MMP-9 concentration as a function of time. The kinetic parametersk cat and K m were obtained by non-linear least squares fitting of the initial rate dependence on the total pro-MMP-9 concentration to the Michaelis-Menten equation using SCIENTIST (MicroMath Scientific Software, Salt Lake City, UT). The values for t 1/2 were calculated from the following general relationship for first-order reactions:k cat × t 1/2 = 0.693. Interactions of latent and active monomer and dimer pro-MMP-9/MMP-9 with TIMP-1 and TIMP-2 were studied using a Fison Iasys™ instrument. TIMP-1 (69 pmol) and TIMP-2 (42 pmol) were immobilized onto activated CM5 sensor cells (Fison), as described (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Under these conditions, 320–380 arc s of TIMP-2 and 310–360 arc s of TIMP-1 were covalently coupled. Binding reactions were carried out essentially as described previously (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The equilibrium constants (K d) were calculated from the rate constants for association (ka) and dissociation (kd) from the equation K d =kd /ka . For biphasic binding K d =k d(2)/k a(1) andk d(1)/k a(2) for the low and high affinity binding sites, respectively. The binding constants for each analyte protein were determined in duplicate using at least six different concentrations of analyte (2–400 nm), in a final volume of 200 μl, where the response increased as a function of analyte concentration. For TIMP-1 and TIMP-2, pro-MMP-9Mand active MMP-9M were titrated from 10 to 125 nm and proMMP-9D and active MMP-9Dwere titrated from 5 to 75 nm. Furthermore, each analyte protein (100 nm) was subjected to analysis using a derivatized sensor cell to determine the amount of nonspecific binding to the carboxymethyl dextran matrix. No binding of the analyte protein to the underivatized matrix was observed. The binding curves were analyzed using the nonlinear data-fitting program Iasys Fastfit™, using both monophasic and biphasic models to obtain the rate constants. Analysis of the data fit the biphasic model, as we have previously described in detail (30.Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). To determine the inhibition constant (K i) of TIMP-1 and TIMP-2 for the active monomer and dimer, the rate constants (k on andk off) were determined under the following conditions, and from these the K i was calculated (i.e. k off/k on). The fluorogenic peptide substrate concentration for each assay was 7 μm, a concentration ∼5-fold greater than the experimentally determined K m for the reaction of the substrate with MMP-9M and MMP-9D. TIMP-1 (0–30 nm) or TIMP-2 (0–60 nm) were added to the fluorogenic substrate solution, and the assay was initiated by addition of enzyme to give a final concentration of 1 nm for MMP-9M and 0.5 nm for MMP-9D. The reaction was allowed to proceed for 6 min, and the rate of substrate cleavage was measured in triplicate for each inhibitor concentration examined. The first-order rate constant, k, was determined from the intersection point of the tangent to the curve atI = x to the curve at I = 0 where k = 1/t, as described (37.Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar), where the data points gave equal increments of product formation as a function of time in the absence of inhibitor. The first-order rate constant,k, for each TIMP concentration was plotted as a function of TIMP concentration. The slope and error of the slope of this line gives the on-rate, k on , as determined by linear regression using LINEST (Microsoft Excel™ version 5.0). The dissociation rate constants (k off) were determined in triplicate as follows. MMP-9M or MMP-9D (300 nm) and inhibitors (330 nm) were incubated for 1 h at 25 °C. These reaction mixtures were added to a cuvette containing 2 ml of a 12 μm peptide substrate solution. The final enzyme concentration was 0.5 nm. The recovery of enzyme activity was followed for up to 40 min, and the data were analyzed as described (38.Glick B.R. Brubacher L.J. Leggett D.J. Can. J. Biochem. 1978; 56: 1055-1057Crossref PubMed Scopus (23) Google Scholar). The error of the slope of this line was determined by linear regression using LINEST (Microsoft Excel™ version 5.0). The inhibition constants (K i) were calculated fromK i =k off/k on. The primary sequence of pro-MMP-9 was obtained from the Swiss-Prot data bank (code COG9_HUMAN, total 707 amino acids). A complete model of pro-MMP-9 was constructed as described below. The signal peptide was removed from the complete sequence, and the remaining sequence was divided into prodomain, catalytic, gelatin-binding, and hemopexin-like domains. Homology models were constructed using the COMPOSER module in Sybyl version 6.4; the modeling of the three-dimensional structure of the catalytic domain of MMP-9 has been published previously (13.Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (695) Google Scholar). The hemopexin-like domain of pro-MMP-9 was modeled using the structures of the hemopexin-like domains of pro-MMP-2 (Protein Data Bank codes 1gen and 1rtg), fibroblast collagenase (code 1fbl), and collagenase-3 (code 1pex), following similar procedures that were used for the modeling of the catalytic domain (13.Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (695) Google Scholar, 39.Libson A.M. Gittis A.G. Collier I.E. Marmer B.L. Goldberg G.I. Lattman E.E. Nat. Struct. Biol. 1995; 2: 938-942Crossref PubMed Scopus (64) Google Scholar, 40.Gohlke U. Gomis-Ruth F.X. Crabbe T. Murphy G. Docherty A.J. Bode W. FEBS Lett. 1996; 378: 126-130Crossref PubMed Scopus (80) Google Scholar, 41.Gomis-Ruth F.X. Gohlke U. Betz M. Knauper V. Murphy G. Lopez-Otin C. Bode W. J. Mol. Biol. 1996; 264: 556-566Crossref PubMed Scopus (116) Google Scholar). The gelatin-binding domain was modeled using the NMR structure of the fibronectin type-II model (code 1fn2) (42.Pickford A.R. Potts J.R. Bright J.R. Phan I. Campbell I.D. Structure. 1997; 5: 359-370Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The prodomain of pro-MMP-9 was modeled based on the x-ray structure of homologous prodomain of stromelysin-1 (code 1slm) (43.Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMe" @default.
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• W1964855331 title "Characterization of the Monomeric and Dimeric Forms of Latent and Active Matrix Metalloproteinase-9" @default.
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