FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2067207214> ?p ?o ?g. }

• W2067207214 endingPage "17874" @default.
• W2067207214 startingPage "17869" @default.
• W2067207214 abstract "An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the Km and kcat values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr63-sulfated hirudin54-65 peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a KD of ∼2 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD > 4-30 μm). Interestingly, an ∼50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom. An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the Km and kcat values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr63-sulfated hirudin54-65 peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a KD of ∼2 μm, it is ineffective in inhibiting the activation of mutant zymogens (KD > 4-30 μm). Interestingly, an ∼50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom. Prothrombin is a vitamin K-dependent serine protease zymogen that can be proteolytically converted to thrombin by factor Xa (FXa) 1The abbreviations used are: FXa, factor Xa; PC, phosphatidylcholine; PS, phosphatidylserine; GPR-pNA, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide; TBS, Tris-buffered saline. 1The abbreviations used are: FXa, factor Xa; PC, phosphatidylcholine; PS, phosphatidylserine; GPR-pNA, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide; TBS, Tris-buffered saline. that has been assembled into the prothrombinase complex (cofactor Va, negatively charged phospholipid vesicles and Ca2+) (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar, 2Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Kettner C. Shaw E. Mann K.G. J. Biol. Chem. 1981; 256: 6537-6540Abstract Full Text PDF PubMed Google Scholar, 4Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (980) Google Scholar, 5Tracy P.B. Rohrbach M.S. Mann K.G. J. Biol. Chem. 1983; 258: 7264-7267Abstract Full Text PDF PubMed Google Scholar). Factor Xa must cleave two peptide bonds at the P1 Arg273 and P1 Arg322 sites to convert prothrombin to thrombin (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar). Although FXa can by itself catalyze the cleavage of both peptide bonds on the substrate, its catalytic efficiency is improved by greater than 5 orders of magnitude when it is assembled into the prothrombinase complex (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (448) Google Scholar, 2Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 3Nesheim M.E. Kettner C. Shaw E. Mann K.G. J. Biol. Chem. 1981; 256: 6537-6540Abstract Full Text PDF PubMed Google Scholar, 4Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (980) Google Scholar, 5Tracy P.B. Rohrbach M.S. Mann K.G. J. Biol. Chem. 1983; 258: 7264-7267Abstract Full Text PDF PubMed Google Scholar). Previous kinetic data have indicated that such a dramatic improvement in the rate of prothrombin activation by the prothrombinase complex is derived from an ∼100-fold decrease in the apparent Km and a greater than 1000-fold enhancement in the kcat of the activation reaction (2Rosing J. Tans G. Govers-Riemslag J.W.P. Zwaal R.F.A. Hemker H.C. J. Biol. Chem. 1980; 255: 274-283Abstract Full Text PDF PubMed Google Scholar, 6Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). In addition to FXa, several activators from different snake venoms have been isolated that can also cleave the two peptide bonds on prothrombin to generate thrombin (7Owen W.G. Jackson C.M. Thromb. Res. 1973; 3: 705-714Abstract Full Text PDF Scopus (79) Google Scholar, 8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 9Morita T. Iwanaga S. Methods Enzymol. 1981; 80: 303-311Crossref Scopus (36) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 11Rao V.S. Swartup S. Kini R.M. Blood. 2003; 102: 1347-1354Crossref PubMed Scopus (46) Google Scholar). One such an activator, which also exhibits FXa-like properties, has been isolated from the venom of Oxyuranus scutellatus scutellatus (taipan venom) (7Owen W.G. Jackson C.M. Thromb. Res. 1973; 3: 705-714Abstract Full Text PDF Scopus (79) Google Scholar, 8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Similar to FXa, it is previously shown that the N terminus of the taipan venom activator contains several γ-carboxyglutamic acid residues that can interact with negatively charged membrane surfaces in the presence of Ca2+ (10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Moreover, similar to FXa, the venom activator is by itself a very poor activator of prothrombin unless it is in complex with a venomic protein cofactor containing an apparent molecular mass of ∼220 kDa (10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Similar to factor Va, the snake venom cofactor dramatically accelerates the activation of prothrombin by the taipan venom activator on negatively charged membrane surfaces (10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar).The mechanism by which factor Va improves the catalytic efficiency of FXa in the prothrombinase complex is not very well understood. However, it was recently demonstrated that the interaction of proexosite-1 of prothrombin with a recognition site on the prothrombinase complex is required for the efficient activation of the substrate (12Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Thus, a proexosite-1-specific peptide ligand derived from the C-terminal domain of the leech inhibitor, hirudin, effectively inhibited the activation of prothrombin by FXa in the presence but not in the absence of factor Va (12Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This observation suggested that factor Va in the prothrombinase complex may provide a binding site for direct interaction with the proexosite-1 on prothrombin (12Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In support of this hypothesis, we showed that the basic residues of proexosite-1 are required for the factor Va-dependent recognition and activation of prothrombin by FXa in the prothrombinase complex (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Thus, the charge reversal mutants of proexosite-1 were activated normally by FXa alone, but their activation by the protease in the presence of factor Va was dramatically impaired (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Further support for a direct interaction between basic residues of proexosite-1 with a complementary site of factor Va was provided by the observation that the mutagenesis of a hirudin-like sequence on the C-terminal heavy chain of factor Va dramatically compromised the cofactor function of factor Va in accelerating the FXa activation of prothrombin by the prothrombinase complex (14Beck D.O. Bukys M.A. Singh L.S. Szabo K.A. Kalafatis M. J. Biol. Chem. 2004; 279: 3084-3095Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).To determine whether, similar to activation by the prothrombinase complex, the basic residues of proexosite-1 constitute a recognition site for interaction with the taipan venom complex, the kinetics of activation of the proexosite-1 charge reversal mutants of prethrombin-1 (prothrombin lacking both the Gla and Kringle-1 domains) by the partially purified venom was studied. The prethrombin-1 mutants in which Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 (chymotrypsinogen numbering (15Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (817) Google Scholar)) were substituted with a Glu in individual constructs were expressed in mammalian cells and purified to homogeneity as described (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The taipan venom activator complex was partially purified by the QAE-Sephadex ion exchange chromatography as described (8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). The SDS-PAGE and a functional activity assays revealed that both the FXa-like activator and the higher molecular weight cofactor of taipan venom are copurified by these methods. The activation kinetic data suggested that, similar to activation by the prothrombinase complex, the Km and kcat values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) have been markedly impaired. Further kinetic studies in the presence of the proexosite-1 specific peptide Tyr63-sulfated hirudin54-65 suggested that the hirudin peptide inhibits the taipan venom activation of prethrombin-1 with a KD of ∼2 μm. However, the competitive inhibitory effect of the hirudin peptide on the activation of the mutants was impaired at varying degrees, which correlated well with the extent of the impairments observed in the activation of mutant zymogens. In agreement with previous results (10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar), the catalytically active subunit of the taipan venom complex was determined to be an ∼50-kDa molecule that could catalyze the rapid activation of prothrombin in a factor Va-dependent manner. Similar to the factor Va-dependent inhibition of FXa, the hirudin peptide inhibited the activation of prothrombin by the isolated venom activator in the presence of factor Va. These results suggest that the basic residues of proexosite-1 are specific recognition sites for a factor Va-like cofactor in the taipan venom complex. The results further suggest that an interaction between the basic proexosite-1 on prothrombin and an acidic region on factor Va accounts for the mechanism of the rate accelerating effect of the cofactor in the prothrombinase complex.EXPERIMENTAL PROCEDURESConstruction and Expression of Mutant Proteins—The expression of wild type prethrombin-1 (prothrombin lacking both γ-carboxyglutamic acid and Kringle-1 domains) by the pNUT-PL2 expression/purification vector system in baby hamster kidney cells has been described previously (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Rezaie A.R. Yang L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12051-12056Crossref PubMed Scopus (45) Google Scholar). Prethrombin-1 mutants in the chymotrypsinogen numbering system (15Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (817) Google Scholar), R35E, K36E, R67E, K70E, R73E, R75E, and R77E, were prepared by PCR mutagenesis methods and expressed by the same vector system as described (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Both the zymogenic and enzymatic properties of mutant proteins have been extensively characterized in previous studies (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Rezaie A.R. Yang L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12051-12056Crossref PubMed Scopus (45) Google Scholar).Human plasma proteins, antithrombin, factor Va, and FXa were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Phospholipid vesicles containing 80% phosphatidylcholine and 20% phosphatidylserine (PC/PS) were prepared as described (17Smirnov M.D. Esmon C.T. J. Biol. Chem. 1994; 269: 816-819Abstract Full Text PDF PubMed Google Scholar). The chromogenic substrates S2238, S2765, and S2222 were purchased from Kabi Pharmacia/Chromogenix (Franklin, OH). The chromogenic substrate N-p-tosyl-Gly-Pro-Arg-p-nitroanilide (GPR-pNA), Tyr63-sulfated hirudin54-65 (Hir54-65(SO−3), and O. scutellatus scutellatus (taipan venom) were purchased from Sigma. Unfractionated heparin (heparin sodium injection, 10,000 units/ml) from beef lung and the active anti-thrombin-binding pentasaccharide fragment of heparin (fondaparinux sodium) were purchased from Quintiles Clinical Supplies (Mt. Laurel, NJ). The prothrombin activating complex was partially purified from the crude venom by an ion exchange chromatography using QAESephadex A-50 (Amersham Biosciences) as described previously (8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Thus, 25 mg of crude venom was dissolved in 2 ml of 0.1 m NaCl and 50 mm NaOAc, pH 5.8, and applied to a QAE-Sephadex ion exchange column (10 × 1 cm) equilibrated with the same buffer. The bound proteins were eluted with a linear gradient of 0.1-0.6 m NaCl using a fast protein liquid chromatography system.SDS-PAGE—Pooled fractions from two QAE-Sephadex A-50 peaks were analyzed on 10% SDS-PAGE under nonreducing conditions. To identify the active subunit of the activator complex, the gel was briefly rinsed with 0.1 m NaCl and 20 mm Tris-HCl, pH 7.5 (TBS), and then overlaid with 1 mm S2222 in 1% low melting agarose. Following 15-30 min of incubation at room temperature, the active subunit of the taipan venom was identified by its ability to cleave the chromogenic substrate S2222 and thus generate a yellow dye in the gel. The gel was then developed with Coomassie Blue. To isolate the active subunit, 100 μg of the partially purified taipan venom was applied on a 10% preparative polyacrylamide gel, and following electrophoresis, the protein band possessing amidolytic activity was sliced out of the gel and transferred to a dialysis bag containing 1 ml of 20 mm Tris-HCl, pH 7.5, and electroeluted at 4 °C. The concentration of the venom activator was determined by stoichiometric titration of the enzyme with a known concentration of antithrombin in complex with an unfractionated high molecular weight heparin as described (18Rezaie A.R. Protein Sci. 1998; 7: 349-357Crossref PubMed Scopus (29) Google Scholar).Prothrombin and Prethrombin-1 Activation—The initial rate of prothrombin and prethrombin-1 activation by taipan venom was studied by incubating different concentrations of the substrate (0.2-30 μm) with QAE-Sephadex purified activator complex (140 pm) in TBS containing 0.1 mg/ml bovine serum albumin, 0.1% polyethylene glycol 8000, and 5 mm CaCl2 (TBS/Ca2+). Following 3-30 min of incubation at room temperature, small aliquots of the activation reactions were transferred to wells of a 96-well assay plate containing 20 mm EDTA, and the rate of thrombin generation was determined from the cleavage of S2238 (200 μm) at 405 nm by a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). The concentration of the generated thrombin was determined from standard curves prepared from the cleavage rate of S2238 (200 μm) by known concentrations of wild type and mutant thrombins as described (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The apparent Km and kcat values for prethrombin-1 activation were calculated from the Michaelis-Menten equation. In all reactions, it was ensured that less than 10% of prethrombin-1 was activated at all concentrations of the substrates.The factor Va-dependent activation of prothrombin by the isolated ∼50 kDa venom activator was utilized to evaluate the affinity of the venom activator for the human cofactor. Thus, the activation of prothrombin (0.5 μm) by the isolated activator (6.5 pm) was monitored on PC/PS vesicles (35 μm) in TBS/Ca2+ as a function of increasing concentrations of human factor Va (0.08-10 nm). Following 3-5 min of incubation at room temperature, EDTA was added to a final concentration of 20 mm, and the rate of thrombin generation was determined by an amidolytic activity assay using S2238 as described above. The kinetics of prothrombin activation by the isolated activator in complex with a saturating concentration of factor Va (30 nm) was also studied as a function of increasing concentrations of prothrombin (7.8-1000 nm) by the same procedures.Prethrombin-1 Activation in the Presence of Hir54-65(SO−3)—The inhibitory effect of the hirudin peptide on the kinetics of prethrombin-1 activation by the taipan venom complex was studied as described (12Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, the rate of activation of each prethrombin-1 derivative (1 μm) by the QAE-Sephadex purified venom (140 pm) was monitored in the presence of increasing concentrations of the hirudin peptide (0-30 μm) in TBS/Ca2+. The concentration of thrombin generated in each reaction was calculated from standard curves as described above except that GPR-pNA was used as the chromogenic substrate. It is known that the cleavage rate of this substrate is not affected by the occupancy of exosite-1 by the hirudin peptide (19Liu L.-W. H. T. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar). The hirudin peptide inhibition of prethrombin-1 activation by the ∼50-kDa isolated active subunit was also monitored in the presence of a saturating concentration of factor Va (30 nm) on PC/PS phospholipid vesicles (35 μm) in TBS/Ca2+. To simplify comparisons of the hirudin peptide dependence of the activation reactions, the data for all activation reactions were normalized to maximal thrombin generation in the absence of the peptide. The dissociation constants (KD) for the interaction of the hirudin peptide with prethrombin-1 derivatives were calculated from Equations 1 and 2 as described (12Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Vobs=(Vlim-Vo)[Pre-1⋅Hir][Pre-1]o+Vo(Eq. 1) [Pre-1⋅Hir]=((KD+[Pre-1]o+[Hir]o)-Sqrt((KD+[Pre-1]o+[Hir]o)2-4[Pre-1]o[Hir]o)2)(Eq. 2) Vobs is the observed initial rate of prethrombin-1 (Pre-1) activation; Pre-1 is prethrombin-1; Vlim is the limiting rate at a saturating hirudin peptide concentration; Hir is hirudin peptide; Vo is the initial rate of activation in the absence of the hirudin peptide; KD is the dissociation constant for the hirudin peptide binding to prethrombin-1; and [Pre-1·Hir] represents the prethrombin-1-hirudin peptide complex concentration.RESULTSExpression and Purification of Recombinant Proteins—Wild type and mutant prethrombin-1 derivatives were expressed in baby hamster kidney cells using the pNUT-PL2 expression/purification vector system as described previously (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). All of the recombinant proteins were purified to homogeneity by an immunoaffinity chromatography using the Ca2+-dependent monoclonal antibody HPC4 as described (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The zymogenic and enzymatic properties of these mutants have been extensively characterized in previous studies (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Rezaie A.R. Yang L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12051-12056Crossref PubMed Scopus (45) Google Scholar). All of the mutants are activated normally by FXa in the absence of a cofactor, and the mutant enzymes, with the exception of K70E, which exhibits an elevated Km value for S2238, cleave the chromogenic substrate with Km and kcat values similar to those by the wild thrombin, suggesting that the mutagenesis has not globally changed the conformation of mutant proteins (13Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). SDS-PAGE analysis indicated that all of the mutant proteins have been purified to homogeneity (data not shown).Purification of the Prothrombin Activator from Taipan Venom—The prothrombin activating complex was partially purified from the crude venom by a QAE-Sephadex ion exchange chromatography as described (8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). The bound proteins were eluted as two major peaks at NaCl concentrations of ∼0.22 and ∼0.35 m (Fig. 1A). Both prothrombin and chromogenic substrate activity assays indicated that only the latter peak contains the active protein. SDS-PAGE analysis indicated that the first peak contains at least two inactive protein bands with apparent molecular masses of below 25 kDa (Fig. 1B, lane 2). The second peak, which contained four or five major protein bands with apparent molecular masses of ∼50-250 kDa (Fig. 1B, lane 1) activated prothrombin rapidly and also exhibited amidolytic activity toward FXa-specific chromogenic substrates. The venom activator hydrolyzed S2765 with Km and kcat values of 0.5 mm and 20 s-1, respectively. Previously, the gel filtration of the crude venom on the Sephadex G-200 followed by a QAE-Sephadex ion exchange chromatography has also fractionated the prothrombin activating complex into four or five similar major protein bands (8Walker F.J. Owen W.G. Esmon C.T. Biochemistry. 1980; 19: 1020-1023Crossref PubMed Scopus (44) Google Scholar, 10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). However, the gel filtration step with the commercial crude venom, prior to the ion exchange chromatography, did not yield any further purification advantage in this study (data not shown).To identify the active subunit of the prothrombin activating complex, SDS-polyacrylamide gel of the QAE-Sephadex isolated proteins was incubated with 1 mm S2222 dissolved in TBS containing 1% agarose. The active subunit was identified by its ability to release p-nitroaniline and was thus identified by the appearance of a yellow band in the gel at the point corresponding to the catalytically active enzyme. As shown in Fig. 1C, the active subunit of the taipan venom activator complex migrated at an apparent molecular mass of ∼50 kDa. This band was sliced out of a preparative gel and transferred to a small dialysis bag (12-14-kDa molecular mass cut-off) containing 1 ml of 20 mm Tris-HCl. The active protein was eluted at 4 °C and concentrated by Centricon (Millipore Corp., Bradford, MA), and its activity toward prothrombin was examined in both the absence and the presence of human factor Va on PC/PS vesicles in TBS/Ca2+. In agreement with previous results (10Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar), the ∼50-kDa activator was by itself a poor activator of prothrombin; however, in the presence factor Va, the activator rapidly activated the substrate to thrombin. The cofactor dependence of prothrombin activation revealed that the activator binds to factor Va with a Kd(app) of 0.34 ± 0.04 nm on PC/PS vesicles in the presence of Ca2+ (Fig. 2A). At a saturating concentration of factor Va (Fig. 2B), the activator activated prothrombin with a Km(app) and kcat values of 60 ± 10 nm and 670 ± 30 nm/min/nm, respectively.Fig. 2Activation of prothrombin by the∼50-kDa subunit of taipan venom in complex with factor Va. A, the apparent dissociation constant for the interaction of the isolated ∼50-kDa subunit with factor Va was determined by incubating the activator (3.8 pm) with prothrombin (100 nm) in the presence of increasing concentrations of human factor Va (0.08-10 nm) and 35 μm PC/PS vesicles in TBS/Ca2+. Following 3-5 min of incubation at room temperature, EDTA was added to a final concentration of 20 mm, and the rate of thrombin generation was determined by an amidolytic activity assay using S2238 as described under “Experimental Procedures.” B, the concentration dependence of prothrombin activation by the ∼50-kDa activator (6.5 pm) in the absence (○) and the presence of 30 nm human factor Va (•) was determined on PC/PS vesicles (35 μm) in TBS/Ca2+. The rate of thrombin generation was determined as described above and fitted to the Michaelis-Menten equation to obtain the Km(app) (60 ± 10 nm) and kcat (670 ± 30 nm/min/nm) values in the presence of factor Va.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reaction with Antithrombin and Active Site Titration of Prothrombin Activator—Noting that the ∼50-kDa activator from taipan venom exhibited FXa-like properties, it was rationalized that the activator may also react with antithrombin. However, the incubation of the isolated ∼50-kDa activator or the taipan venom activation complex partially purified by the ion exchange chromatography with 1 μm serpin for 1 h at room temperature did not result in a significant decline in the amidolytic or the proteolytic activities of enzymes. Nevertheless, it was discovered that both the isolated and the activator complex react with the serpin in the presence of a full-length heparin with second order association rate constants of 4.8 ± 0.5 × 103m-1 s-1, thus making it possible to reliably determine the concentration of the active site of the venom activators by their stoichiometric titration with known concentrations of anti-thrombin. It should be noted that SDS-PAGE analysis of the inhibition reactions indicated that, similar to FXa, the venom activator forms a stable high molecular weight complex with the serpin with no evidence for the enzyme recognizing the serpin as a substrate. Further study revealed that the rate accelerating effect of heparin is not mediated through a conformational activation of antithrombin but through a t" @default.
• W2067207214 created "2016-06-24" @default.
• W2067207214 creator A5019234325 @default.
• W2067207214 creator A5078143614 @default.
• W2067207214 date "2004-04-01" @default.
• W2067207214 modified "2023-09-29" @default.
• W2067207214 title "Proexosite-1-dependent Recognition and Activation of Prothrombin by Taipan Venom" @default.
• W2067207214 cites W1483113783 @default.
• W2067207214 cites W1518197977 @default.
• W2067207214 cites W1522112651 @default.
• W2067207214 cites W1537431414 @default.
• W2067207214 cites W1538941022 @default.
• W2067207214 cites W1582785412 @default.
• W2067207214 cites W1835872178 @default.
• W2067207214 cites W1961597147 @default.
• W2067207214 cites W2022755980 @default.
• W2067207214 cites W2041349540 @default.
• W2067207214 cites W2057496200 @default.
• W2067207214 cites W2059718682 @default.
• W2067207214 cites W2060781560 @default.
• W2067207214 cites W2065441127 @default.
• W2067207214 cites W2066902897 @default.
• W2067207214 cites W2067591369 @default.
• W2067207214 cites W2091629675 @default.
• W2067207214 cites W2113691948 @default.
• W2067207214 cites W2141373630 @default.
• W2067207214 cites W2153423854 @default.
• W2067207214 cites W2168328536 @default.
• W2067207214 cites W2170055916 @default.
• W2067207214 cites W32832107 @default.
• W2067207214 cites W998538056 @default.
• W2067207214 doi "https://doi.org/10.1074/jbc.m314285200" @default.
• W2067207214 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14769787" @default.
• W2067207214 hasPublicationYear "2004" @default.
• W2067207214 type Work @default.
• W2067207214 sameAs 2067207214 @default.
• W2067207214 citedByCount "7" @default.
• W2067207214 countsByYear W20672072142015 @default.
• W2067207214 countsByYear W20672072142019 @default.
• W2067207214 crossrefType "journal-article" @default.
• W2067207214 hasAuthorship W2067207214A5019234325 @default.
• W2067207214 hasAuthorship W2067207214A5078143614 @default.
• W2067207214 hasBestOaLocation W20672072141 @default.
• W2067207214 hasConcept C153180895 @default.
• W2067207214 hasConcept C154945302 @default.
• W2067207214 hasConcept C185592680 @default.
• W2067207214 hasConcept C2779448229 @default.
• W2067207214 hasConcept C41008148 @default.
• W2067207214 hasConcept C55493867 @default.
• W2067207214 hasConcept C86803240 @default.
• W2067207214 hasConceptScore W2067207214C153180895 @default.
• W2067207214 hasConceptScore W2067207214C154945302 @default.
• W2067207214 hasConceptScore W2067207214C185592680 @default.
• W2067207214 hasConceptScore W2067207214C2779448229 @default.
• W2067207214 hasConceptScore W2067207214C41008148 @default.
• W2067207214 hasConceptScore W2067207214C55493867 @default.
• W2067207214 hasConceptScore W2067207214C86803240 @default.
• W2067207214 hasIssue "17" @default.
• W2067207214 hasLocation W20672072141 @default.
• W2067207214 hasOpenAccess W2067207214 @default.
• W2067207214 hasPrimaryLocation W20672072141 @default.
• W2067207214 hasRelatedWork W1966390022 @default.
• W2067207214 hasRelatedWork W1985478747 @default.
• W2067207214 hasRelatedWork W2054082043 @default.
• W2067207214 hasRelatedWork W2600658134 @default.
• W2067207214 hasRelatedWork W2609838162 @default.
• W2067207214 hasRelatedWork W2748952813 @default.
• W2067207214 hasRelatedWork W2899084033 @default.
• W2067207214 hasRelatedWork W2997499382 @default.
• W2067207214 hasRelatedWork W3013367349 @default.
• W2067207214 hasRelatedWork W3119253307 @default.
• W2067207214 hasVolume "279" @default.
• W2067207214 isParatext "false" @default.
• W2067207214 isRetracted "false" @default.
• W2067207214 magId "2067207214" @default.
• W2067207214 workType "article" @default.