Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m108422200
ISSN1083-351X
AutoresJayeeta Dhar, Abhay H. Pande, Vasudha Sundram, Jagpreet S. Nanda, Shekhar C. Mande, Girish Sahni,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe selective deletion of a discrete surface-exposed epitope (residues 254–262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254–262). A kinetic analysis of SKdel254–262 revealed that its low HPG activator activity arose from a 5–6-fold increase in Kmfor HPG as substrate, with little alteration in kcatrates. This increase in the Kmfor the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SKdel254–262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254–262· plasmin activator complex to ε-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK·plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1–5 of HPG to SKdel254–262·HPG was reduced by about 3-fold in comparison with that of nSK·HPG. Overall, these observations identify the 250 loop in the β domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK·HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process. The selective deletion of a discrete surface-exposed epitope (residues 254–262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254–262). A kinetic analysis of SKdel254–262 revealed that its low HPG activator activity arose from a 5–6-fold increase in Kmfor HPG as substrate, with little alteration in kcatrates. This increase in the Kmfor the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SKdel254–262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254–262· plasmin activator complex to ε-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK·plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1–5 of HPG to SKdel254–262·HPG was reduced by about 3-fold in comparison with that of nSK·HPG. Overall, these observations identify the 250 loop in the β domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK·HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process. streptokinase plasminogen (irrespective of source) human plasminogen human plasmin microplasmin microplasminogen kringles 1–5 of plasminogen p-nitrophenyl p-guanidinobenzoate splicing-overlap-extension ε-amino caproic acid native-like SK Streptokinase (SK),1 a bacterial protein secreted by the Lancefield Group C β-hemolytic streptococci, is widely used as a thrombolytic agent in the treatment of various circulatory disorders, including myocardial infarction (1.International Study of Infarct Survival-3Lancet. 1992; 339: 753-781Abstract PubMed Scopus (1145) Google Scholar). Unlike other human plasminogen (HPG) activators, like tissue plasminogen activator and urokinase, SK does not possess any intrinsic enzymatic activity. Instead, SK forms an equimolar, stoichiometric complex with “partner” HPG or plasmin (HPN), which then catalytically activates free “substrate” molecules of HPG to HPN by selective cleavage of the Arg561-Val562 peptide bond (2.Castellino F.J. Chem. Rev. 1981; 81: 431-446Crossref Scopus (122) Google Scholar, 3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar). It is believed that consequent to the initial SK·HPG complexation, there is a structural rearrangement within the complex, and even before any proteolytic cleavage takes place, an active center within the HPG moiety capable of undergoing acylation is formed (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar). This activated complex is rapidly transformed into an SK·HPN complex and develops an HPG activator activity. Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK·HPN displays a very narrow substrate specificity (4.Markus G. Werkheiser W.C. J. Biol. Chem. 1964; 239: 2637-2643Abstract Full Text PDF PubMed Google Scholar). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the “cofactor” SK, with exclusive propensity for acting on the target scissile peptide bond in HPG has been the subject of active investigations with both fundamental and applied implications (5.Parrado J. Conejero-Lara F. Smith R.A.G. Marshall J.M. Ponting C.P. Dobson C.M. Protein Sci. 1996; 5: 693-704Crossref PubMed Scopus (53) Google Scholar, 6.Esmon C.T. Mather T. Nat. Struct. Biol. 1998; 5: 933-937Crossref PubMed Scopus (27) Google Scholar, 7.Conejero-Lara F. Parrado J. Azuaga A.I. Smith R.A.G. Ponting C.P. Dobson C.M. Protein Sci. 1996; 5: 2583-2591Crossref PubMed Scopus (27) Google Scholar, 8.Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar, 9.Parry M.A. Zhang X.C. Bode W. Trends Biochem. Sci. 2000; 25: 53-59Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10.Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11.Nihalani D. Raghava G.P.S. Sahni G. Protein Sci. 1997; 6: 1284-1292Crossref PubMed Scopus (27) Google Scholar, 12.Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Crossref PubMed Scopus (41) Google Scholar). SK has been shown to be composed of three structurally similar domains (termed α, β, and γ), separated by random coils and small, flexible regions at the amino and carboxyl termini (5.Parrado J. Conejero-Lara F. Smith R.A.G. Marshall J.M. Ponting C.P. Dobson C.M. Protein Sci. 1996; 5: 693-704Crossref PubMed Scopus (53) Google Scholar, 7.Conejero-Lara F. Parrado J. Azuaga A.I. Smith R.A.G. Ponting C.P. Dobson C.M. Protein Sci. 1996; 5: 2583-2591Crossref PubMed Scopus (27) Google Scholar, 8.Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar). The recently solved crystal structure of the catalytic domain of HPN complexed with SK strongly indicates how SK might modulate the substrate specificity of HPN by providing a “valley” or cleft in which the macromolecular substrate can dock through protein-protein interactions, thus positioning the scissile peptide bond optimally for cleavage by the HPN active site, thereby conferring a narrow substrate preference onto an otherwise “indiscriminate” active center. In this structure, SK does not appear to induce any significant conformational changes in the active site residues directly but, along with partner HPG, seems to provide a template on which the substrate molecule can dock through protein-protein interactions, resulting in the optimized presentation of the HPG activation loop at the active center of the complex (6.Esmon C.T. Mather T. Nat. Struct. Biol. 1998; 5: 933-937Crossref PubMed Scopus (27) Google Scholar, 8.Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar). However, the identity of these interactions and their contributions to the formation of the enzyme-substrate intermediate(s) remains a mystery so far. Besides the well recognized “switch” in substrate preference (4.Markus G. Werkheiser W.C. J. Biol. Chem. 1964; 239: 2637-2643Abstract Full Text PDF PubMed Google Scholar), the binding of SK to HPN results in a severalfold enhancement of the Km for diverse small molecular weight chromogenic peptide substrates but relatively little alteration in their kcat values as compared with free HPN, indicating that the primary, covalent specificity characteristics of the active center of HPN upon SK binding are unchanged but result in steric hindrance/reduced accessibility for even the small molecular weight peptide substrates. Thus, the remarkable alteration of the macromolecular substrate specificity of HPN by SK is currently thought to be due to “exosites” generated on the SK·HPN complex, as shown recently by the elegant use of active site-labeled fluorescent HPN derivatives (10.Boxrud P.D. Fay W.P. Bock P.E. J. Biol. Chem. 2000; 275: 14579-14589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Peptide walking studies in our laboratory had also indicated that short peptides based on the primary structure of SK, particularly those derived from selected regions in the α and β domains, displayed competitive inhibition for HPG activation by the preformed SK·HPN complex under conditions where the 1:1 complexation of SK and HPN was essentially unaffected (11.Nihalani D. Raghava G.P.S. Sahni G. Protein Sci. 1997; 6: 1284-1292Crossref PubMed Scopus (27) Google Scholar, 12.Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Crossref PubMed Scopus (41) Google Scholar) However, the crystal structure of SK complexed with microplasmin(ogen) (8.Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar), while providing a high degree of resolution of the residues involved in the SK·μPN complexation, yielded few unambiguous insights regarding the interactions engendered between the activator complex and substrate HPG. This is probably due to the binary nature of the complex (i.e. an absence of a juxtaposed substrate molecule, large average thermal factors especially in the β domain, and a total absence, in both partner and substrate HPG, of the kringles that are known to be important in HPG activation) (13.Lin L-F. Houng A. Reed G.L. Biochemistry. 2000; 39: 4740-4745Crossref PubMed Scopus (33) Google Scholar). Thus, despite a detailed and high resolution exposition of the overall nature of protein-protein interactions in the SK·μPN binary complex, discrete structures/epitopes of SK, if any, that are directly involved in the exosite formation process by the full-length activator complex have not yet been identified. Previously, charged side chains, both in HPG, particularly around the active center (14.Dawson K.M. Marshall J.M. Raper R.H. Gilbert R.J. Ponting C.P. Biochemistry. 1994; 33: 12042-12047Crossref PubMed Scopus (20) Google Scholar), and in SK in the β domain (16.Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar, 17.Lin L-F. Oeun S. Houng A. Reed G.L. Biochemistry. 1996; 35: 16879-16885Crossref PubMed Scopus (34) Google Scholar), have been shown to be important for HPG activation ability. However, a clear cut identification of a structural epitope/element in conferring substrate HPG affinity onto the SK·PG activator complex has not been demonstrated until now. Of the three domains of SK, the central β domain displays maximal affinity for HPG (15.Conejero-Lara F.C. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Crossref PubMed Scopus (40) Google Scholar), the N-terminal α domain displays relatively lesser affinity for HPG with the γ domain showing much less affinity of for HPG. 2V. Sundram, K. Rajagopal, A. Chaudhary, S. S. Komath, and G. Sahni, unpublished observations. Solution and structural studies suggest that both α and β domains are involved in the substrate recognition phenomenon (8.Wang X. Lin X. Loy J.A. Tang J. Zhang X.C. Science. 1998; 281: 1662-1665Crossref PubMed Scopus (219) Google Scholar, 12.Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Crossref PubMed Scopus (41) Google Scholar). Mutagenesis studies have also implicated positively charged residues in the β domain to be important but have failed to show that these residues are directly involved in substrate recognition by the binary complex (17.Lin L-F. Oeun S. Houng A. Reed G.L. Biochemistry. 1996; 35: 16879-16885Crossref PubMed Scopus (34) Google Scholar). An examination of the crystal structure of the free β domain (18.Wang X. Tang J. Hunter B. Zhang X.C. FEBS Lett. 1999; 459: 85-89Crossref PubMed Scopus (33) Google Scholar) and its comparison with the other two SK domains possessing closely similar (but not identical) structures revealed the presence of a distinct flexible loop in the β domain (the 250-loop) that protrudes into the solvent (Fig. 1). In the present study, we chose to delete this loop based on the premise that if this structural motif is involved in substrate recognition, discrete deletion of this loop would lead to a selective increase in Km of the activator complex. The results obtained provide clear cut evidence of the role played by this nine-residue loop in substrate recognition and thus identify a functionally important component of the macromolecular substrate-specific exosite operative in the SK·HPN complex, which interacts via the kringle(s) in HPG. Glu-plasminogen was either purchased from Roche Diagnostics Inc. or purified from human plasma by affinity chromatography (19.Deutsch D.G. Mertz E.T. Science. 1970; 170: 1095-1096Crossref PubMed Scopus (1670) Google Scholar). The RNA polymerase promoter-based expression vector, pET23(d) and Escherichia coli strain BL21 (DE3) were products of Novagen Inc. (Madison, WI). Thermostable DNA polymerase (pfu) was obtained from Stratagene Inc. (La Jolla, CA), and restriction endonucleases, T4 DNA ligase, and other DNA-modifying enzymes were acquired from New England Biolabs (Beverly, MA). Oligonucleotide primers were supplied by Integrated DNA Technologies Inc., (Indianapolis, IN). HPN was prepared by digesting Glu-HPG with urokinase covalently immobilized on agarose beads using a ratio of 300 Plough units/mg HPG in 50 mm Tris-Cl, pH 8.0, 25% glycerol, and 25 mml-lysine at 22 °C for 10 h (15.Conejero-Lara F.C. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Crossref PubMed Scopus (40) Google Scholar, 16.Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar). All other reagents were of the highest analytical grade available. A set of mutagenic and flanking primers, carrying unique restriction sites, were used in polymerase chain reactions to generate DNA fragments having overlapping ends. Thereafter, splicing-overlap-extension PCR (SOE-PCR) reactions (20.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6832) Google Scholar) were carried out, resulting in amplification products, which were cloned in the pET23(d) expression vector (16.Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar, 21.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar). Oligonucleotide primers for SOE-PCR for the construction of SKdel254–262 were as follows. The mutagenic primers were as follows: upstream primer, 5′-AACAGGCTTATAGGGAAATAAACAACACTGACCTGATATCTGAGAAA-3′; downstream primer, 5′ TGTTGTTTATTTCCCTATAAGCCTGTTCCCGATTTTTAA 3′. The flanking primers were as follows: upstream primer, 5′-ATTTATGAACGTGACTCCTCAATCGTC-3′; downstream primer, 5′-ATAGGCTAAATGATAGCTAGCATTCTCTCC-3′. Oligonucleotide primers for SOE-PCR for the construction of βwild type and βdel254–262 were as follows. Sequences of the mutagenic primers were as follows: upstream primer, 5′-AACAGGCTTATAGGGAAATAAACAACACTGACCTGATATCTGAGAAA-3′; downstream primer, 5′-TGTTGTTTATTTCCCTATAAGCCTGTTCCCGATTTTTAA-3′. Sequences of the flanking primers were as follows: upstream primer, 5′-GTGGAATATACTGTACAGTTTACTCC-3′; downstream primer, 5′-ATCGGGATCCTATTTCAAGTGACTGCGATCAAAGGG-3′. Both proteins were expressed intracellularly in E. coli BL21 (DE3) cells after induction with isopropyl-1-thio-β-d-galactopyranoside essentially according to the instructions of the supplier (Novagen Inc.). The host-vector system for the expression of the cDNA corresponding to mature SK from Streptococcus equisimilis H46A after in-frame juxtaposition of an initiator methionine codon (so as to express the protein as Met-SK) has been described earlier (16.Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar). However, protein sequence analysis of the purified SK expressed intracellularly in E. coli (referred to as nSK hereafter) was found to have its N-terminal Met removed at a 50% level. The same case was seen in the mutant (SKdel254–262) prepared similarly from E. coli employing the same expression vector. The pelleted cells were sonicated, and the proteins in the supernatants were precipitated with ammonium sulfate (16.Chaudhary A. Vasudha S. Rajagopal K. Komath S.S. Garg N. Yadav M. Mande S.C. Sahni G. Protein Sci. 1999; 8: 2791-2805Crossref PubMed Scopus (38) Google Scholar). This fraction, after dissolution in 20 mm Tris-Cl buffer, pH 7.5, was then chromatographed on a Poros-D anion exchange column fitted onto a Bio-Cad Sprint liquid chromatographic workstation (Perseptive Biosystems Inc., Framingham, MA). nSK/SKdel254–262 were eluted using a linear gradient of NaCl (0–0.5 m) in 20 mm Tris-Cl buffer, pH 7.5. The eluted proteins were more than 95% pure, as analyzed by SDS-PAGE. Both proteins were expressed intracellularly in E. coli BL21 (DE3) cells as inclusion bodies. The pellet obtained after sonication was taken up in 8 m urea and placed under gentle shaking conditions for 30 min to effect dissolution. After a high speed centrifugation step, the protein in the supernatant was refolded by 20-fold dilution with 20 mm Tris-Cl buffer, pH 7.5. The β domain was then purified to more than 95% homogeneity by chromatography on DEAE-Sepharose Fast-flow (Amersham Biosciences) at 4 °C using a linear NaCl gradient (0–0.25 m NaCl in 20 mm Tris-Cl buffer, pH 7.5). Microplasminogen, the catalytic domain of plasminogen (residues Lys530–Asn790) devoid of all kringles was prepared by cleavage of HPG by HPN under alkaline conditions (0.1 n glycine/NaOH buffer, pH 10.5) at 25 °C. Microplasminogen was purified from the reaction mixture by passing through a Lys-Sepharose column (Amersham Biosciences), followed by a soybean-trypsin inhibitor-Sepharose 4B column to absorb HPN and μPN, as reported (22.Shi G-Y. Wu H-L. J. Biol. Chem. 1988; 263: 17071-17075Abstract Full Text PDF PubMed Google Scholar). The flow-through was then subjected to molecular sieve chromatography, after concentration by ultrafiltration, on a column (16 × 60 cm) of Superdex-75TM (Amersham Biosciences). The purity of μPG formed was analyzed by SDS-PAGE, which showed a single band moving at the position expected from its molecular size (22.Shi G-Y. Wu H-L. J. Biol. Chem. 1988; 263: 17071-17075Abstract Full Text PDF PubMed Google Scholar). The proteolytic fragment containing all of the HPG kringle domains (K1–5) was prepared by incubating HPG with urokinase-free HPN (5:1 ratio of HPG and HPN) under alkaline conditions (0.1 n glycine/NaOH, pH 9.0) for 72 h at 25 °C. Under these conditions, the proteolytic conversion of native HPG to K1–5 was found to be quantitative, with minimal residual HPG. HPN was removed from the reaction mixture by passing through a soybean trypsin inhibitor-Sepharose column (1.6 × 3.6 cm). This was followed by gel filtration on Superdex-75, to obtain HPG- and μPG-free K1–5. The purity of this preparation was confirmed by SDS-PAGE analysis (23.Wu H.-L. Chang B.-I. Wu D.-H. Chang L.-C. Gong G.-C. Lou K.-L. Shi G.-Y. J. Biol. Chem. 1990; 265: 19658-19664Abstract Full Text PDF PubMed Google Scholar). Activation with urokinase, which is known to be a good activator of μPG irrespective of the presence of kringle domains (22.Shi G-Y. Wu H-L. J. Biol. Chem. 1988; 263: 17071-17075Abstract Full Text PDF PubMed Google Scholar), was used to establish that the activation of this preparation, when used as substrate, was comparable with that obtained when using SK·HPN as the activator species. Aliquots (50 nm) were withdrawn from equimolar HPG·nSK/SKdel254–262 complexes at regular periods and transferred to a 100-μl quartz microcuvette containing 2 mm tosyl-glycyl-prolyl-lysine-4-nitranilide-acetate (Chromozym® PL) and 50 mm Tris-Cl, pH 7.5, at 22 °C. The change in absorbance at 405 nm was monitored to compute the kinetics of amidolytic activation (12.Nihalani D. Kumar R. Rajagopal K. Sahni G. Protein Sci. 1998; 7: 637-648Crossref PubMed Scopus (41) Google Scholar, 24.Wohl R.C. Summaria L. Robbins K.C. J. Biol. Chem. 1980; 255: 2005-2013Abstract Full Text PDF PubMed Google Scholar). Five μm HPG was added to an assay cuvette containing 5.5 μm nSK/SKdel254–262, 100 μmNPGB, and 10 mm phosphate buffer, pH 7.5, and the “burst” of p-nitrophenol release was monitored at 410 nm as a function of time at 22 °C (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 25.Chase Jr., T. Shaw E. Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (380) Google Scholar). Varying amounts of HPG were added to the assay cuvette containing fixed amounts of nSK/SKdel254–262 and chromogenic substrate (1 mm), and the change in absorbance was monitored at 405 nm as a function of time at 22 °C. Also, the kinetics of HPG activation by HPN·nSK/SKdel254–262 complexes were measured by transferring suitable aliquots of preformed HPN·nSK/SKdel254–262 complexes to the assay cuvette containing different concentrations of substrate HPG (24.Wohl R.C. Summaria L. Robbins K.C. J. Biol. Chem. 1980; 255: 2005-2013Abstract Full Text PDF PubMed Google Scholar). To compute the kcat, the number of HPN active sites was determined using the NPGB reaction (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, 25.Chase Jr., T. Shaw E. Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (380) Google Scholar, 26.Wohl R.C. Arzadon L. Summaria L. Robbins K.C. J. Biol. Chem. 1977; 252: 1141-1147Abstract Full Text PDF PubMed Google Scholar). nSK/SKdel254–262 and HPN were precomplexed at 4 °C in equimolar ratios (100 nm each) for 1 min in 50 mm Tris-Cl, pH 7.5, containing 0.5% bovine serum albumin, and an aliquot of the reaction mixture was transferred to a 100-μl assay cuvette containing 50 mm Tris-Cl buffer, pH 7.5, and varying concentrations of the chromogenic substrate (0.1–2 mm) to obtain a final concentration of 10 nm complex in the reaction. The reaction was monitored spectrophotometrically at 405 nm for 5 min at 22 °C. The kinetic constants were calculated by standard methods (25.Chase Jr., T. Shaw E. Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (380) Google Scholar). Association and dissociation between HPG and the nSK/SKdel254–262, referred to hereafter as binary interaction, were followed in real time by resonant mirror-based detection using the IAsys PlusTMsystem (Cambridge, UK) (27.Cush R. Cronin J.M. Stewart W.J. Maule C.H. Molloy J. Goddard N.J. Biosensors Bioelectronics. 1993; 8: 347-354Crossref Scopus (384) Google Scholar, 28.Buckle P.E. Davies R.J. Kinning T. Yeung D. Edwards P.R. Pollard-Knight D. Lowe C.R. Biosensors Bioelectronics. 1993; 8: 355-363Crossref Scopus (170) Google Scholar). In these experiments, streptavidin was captured on biotin cuvette according to the manufacturer's protocols (IAsys protocol 1.1). This was followed by the attachment of (mildly) biotinylated HPG to the streptavidin captured on the cuvette. Nonspecifically bound HPG was then removed by repeated washing with phosphate-buffered saline followed by three washes with 10 mm HCl. The net response chosen for the immobilized biotinylated HPG onto the cuvette was 700–800 arc seconds in all experiments. Experiments were performed at 25 °C in 10 mm phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20 and 5 × 10−3m NPGB (binding buffer). The latter was included in order to prevent plasmin-mediated proteolysis. After equilibrating the cuvette with binding buffer, varying concentrations of either nSK or SKdel254–262 were added, and each binding response was monitored during the “association” phase. Subsequently, the cuvette was washed with binding buffer, and the “dissociation” phase was recorded (29.Myszka D.G. Curr. Opin. Biotechnol. 1997; 8: 50-57Crossref PubMed Scopus (427) Google Scholar). Following each cycle of analysis, the cuvette was regenerated by washing with 10 mmHCl, and base line was reestablished with binding buffer. In parallel, in the control cell in the dual channel cuvette, immobilized streptavidin alone was taken as a negative control for the binding studies. In experiments where EACA was used to examine its effect on SK-HPG interaction, the binary complex was formed between ligate nSK/SKdel254–262 and immobilized HPG in binding buffer (as described above). The dissociation of the binary complexes was done by washing the cuvette with EACA instead of buffer alone. The data were analyzed after subtraction of the corresponding nonspecific refractive index component(s), and the kinetic constants were calculated from the sensorgrams by nonlinear fitting of the association and dissociation curves according to 1:1 model A+ B = AB using the software FASTfitTM, supplied by the manufacturers. Briefly, the association curves at each concentration of ligate were fitted to the pseudo-first order equation to calculate the observed rate constant (kon). Then the concentration dependence of kon was fitted using linear regression to find the association rate constant (ka) from the slope of the linear fit (30.Morton T.A. Myszka D.G. Chaiken I.M. Anal. Biochem. 1995; 227: 176-185Crossref PubMed Scopus (298) Google Scholar). The dissociation rate constant (kd) was calculated from the average of four dissociation curves obtained at saturating concentration of ligate. The equilibrium dissociation constant (KD) was then calculated as kd/ka. Values of KD obtained using this relationship were in good approximation to those obtained by Scatchard analysis of the extent of association (data not shown). Resonant mirror technology-based biosensor was also used to measure the rate and equilibrium dissociation constants describing interactions between soluble ligate (PG, μPG, or K1–5) and nSK/SKdel254–262complexed with immobilized HPG, a situation simulating substrate binding to binary complex and hereafter referred to as ternary interaction. In binary interaction studies, it was evident that when soluble nSK/SKdel254–262 was added to immobilized HPG, a rapid and avid SK·HPG binary complex formation occurs. The dissociation of this complex is very slow due to the high stability of the SK·HPG complex, as has been observed by others also (15.Conejero-Lara F.C. Parrado J. Azuaga A.I. Dobson C.M. Ponting C.P. Protein Sci. 1998; 7: 2190-2199Crossref PubMed Scopus (40) Google Scholar). After allowing the complex to dissociate maximally (∼20 min), the dissociation base line becomes stable, which remains unaffected even after washing with 2.5 mm EACA. It has been reported that when SK was preincubated with immobilized HPG, EACA was >100-fold less potent at dissociating the binary complex than it was at preventing binary complex formation when SK and EACA were added synchronously to immobilized HPG (13.Lin L-F. Houng A. Reed G.L. Biochemistry. 2000; 39: 4740-4745Crossref PubMed Scopus (33) Google Scholar). Thus, this comparative resistance to dissociation of the SK·HPG binary complex by EACA permitted us to study ternary substrate interaction under conditions that did not adversely affect the stability of the binary interaction. In contrast, EACA was found to be strongly inhibitory to ternary complex formation (see below). In a typical ternary interaction experiment, a stable binary complex was formed by adding a saturating amount of either nSK or SKdel254–262 onto HPG, immobilized on streptavidin captured on biotin cuvette. After maximally dissociating the binary complex with binding buffer and washing with 2.5 mm EACA, a stable dissociation base line was obtained. Varying concentrations of either “ternary” HPG (0.1–1.0 μm), μPG (1–6 μm), or K1–5 (1–6 μm) were then added to monitor the binding by recording the association phase. Subsequently, the cuvette was washed with binding buffer three times, and the dissociation phase was recorded. After each cycle of analysis, the original base line was reestablished by stripping off the undissociated ternary ligate with 2.5 mm EACA followed by three washes with binding buffer. It was established that EACA, at this concentrati
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