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• W2028776941 abstract "Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor Xa by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482–7491). In the present study, we locate three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor Xa. Our results demonstrate that the structural responses of human and bovine factor Xa to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor Xa are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the γ-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca2+ (kd ∼ 1 mm), although this PS site does not influence the functional response of factor Xa. Second, a Ca2+-dependent binding site is in the epidermal growth factor domains (EGFNC) that are linked by Ca2+ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor Xa. Third, a Ca2+-requiring site seems to be in the EGFC-catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of Ca2+, meaning that PS regulation of factor Xa involves linkage between widely separated parts of the protein. Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor Xa by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482–7491). In the present study, we locate three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor Xa. Our results demonstrate that the structural responses of human and bovine factor Xa to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor Xa are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the γ-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca2+ (kd ∼ 1 mm), although this PS site does not influence the functional response of factor Xa. Second, a Ca2+-dependent binding site is in the epidermal growth factor domains (EGFNC) that are linked by Ca2+ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor Xa. Third, a Ca2+-requiring site seems to be in the EGFC-catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of Ca2+, meaning that PS regulation of factor Xa involves linkage between widely separated parts of the protein. 1,2-dicaproyl-sn-glycero-3-phospho-l-serine 1,2-dicaproyl-sn-glycero-3-phosphocholine phosphatidylserine circular dichroism ellipticity ratio at wavelength 222 to 208 nm Russel's viper venom factor X-activating protein polyethylene glycol N-α-benzyloxycarbonyl-d-arginyl-l-glycyl-l-arginine-p-nitroanilide dihydrochloride Spectrozyme PCa N-terminus γ-carboxyglutamic acid-rich region epidermal growth factor nearest to the N terminus epidermal growth factor nearest the C terminus Gla domain linked to the epidermal growth factor EGFN Gla domain linked to both epidermal growth factors EGFN and EGFC factor Xa construct lacking both the Gla and the EGFN domains [5-(dimethylamino)-1-napthalenesulfonyl]glutamylycylarginyl chloromethyl ketone factor Xa construct missing the Gla domain GDFXa mutant in which Tyr-99 is replaced with Thr critical micelle concentration The substantial effects of soluble phosphatidylserine (C6PS1) on the kinetics of prothrombin activation by factor Xa (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar) and on the structure of factor Xa, as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles (2Sandberg H. Bode A.P. Dombrose F.A. Hoechli M. Lentz B.R. Thromb. Res. 1985; 39: 63-79Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 3Sims P.J. Faioni E.M. Wiedmer T. Shattil S.J. J. Biol. Chem. 1988; 263: 18205-18212Abstract Full Text PDF PubMed Google Scholar). The implication of this PS exposure and of the effect of PS on factor Xa and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation. To better define this regulatory process, it is important to know the locations of the PS binding sites on factor Xa. The organization of factor X into structural domains is illustrated below in Fig. 1. Factor X consists of two peptides. The light chain consists of an N terminus γ-carboxyglutamic acid-rich region (Gla module) and two Cys-rich cassette modules. The heavy chain consists of the serine protease catalytic domain. The two cassette modules of the light chain show strong sequence and structural homology to epidermal growth factor (EGF) (4Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar) and are thus referred to as EGFN and EGFC, where N and C indicate the domain nearer to the N and C termini, respectively. Crystal structures of Gla domain-less factor Xa (GDFXa) have been published (5Padmanabhan K. Padmanabhan K.P. Tulinsky A. Park C.H. Bode W. Huber R. Blankenship D.T. Cardin A.D. Kisiel W. J. Mol. Biol. 1993; 232: 947-966Crossref PubMed Scopus (401) Google Scholar, 6Brandstetter H. Kuhne A. Bode W. Huber R. von der Saal W. Wirthensohn K. Engh R.A. J. Biol. Chem. 1996; 271: 29988-29992Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 7Kamata K. Kawamoto H. Honma T. Iwama T. Kim S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6630-6635Crossref PubMed Scopus (121) Google Scholar). In the most recent of these (7Kamata K. Kawamoto H. Honma T. Iwama T. Kim S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6630-6635Crossref PubMed Scopus (121) Google Scholar), the EGF cassette modules extend from the catalytic domain to make an extended molecule. In the structure of the analogous serine protease, factor IXa, the EGFN module is bent at the inner-EGFC hinge region to right angles with the EGFC, which is tucked along the catalytic module (8Brandstetter H. Bauer M. Huber R. Lollar P. Bode W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9796-9800Crossref PubMed Scopus (261) Google Scholar). It may be that the EGF modules form a hinge region that modulates the global structure of factor Xa. The factor IXastructure also differs from that of GDFXa in containing the Gla domain. Although the Gla domain is critical for membrane binding and may modulate the structure of the EGF modules, little is known about the structure of Gla in whole factor Xa. We have only a model structure of factor Xa Gla domain based on the prothrombin Gla domain (9Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Binding of Ca2+ to factor X is reportedly required for activation by factor VIIa/tissue factor or by factor IXa/VIIIa (10Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Crossref PubMed Scopus (75) Google Scholar, 11Fujimura H. Kambayash J. Monden M. Kato H. Miyata T. Thromb. Haemost. 1995; 74: 1381-1382Crossref PubMed Scopus (50) Google Scholar) and for the activity of factor Xa (12Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (246) Google Scholar). Ca2+ binding is also required for PS regulation of factor Xa proteolytic activity (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). Ca2+ binds mainly to the Gla module (13Henriksen R.A. Jackson C.M. Arch. Biochem. Biophys. 1975; 170: 149-159Crossref PubMed Scopus (63) Google Scholar), but there also appears to be a high affinity Ca2+ binding site (kd ∼ 160 μm) in the catalytic domain (9Sabharwal A.K. Padmanabhan K. Tulinsky A. Mathur A. Gorka J. Bajaj S.P. J. Biol. Chem. 1997; 272: 22037-22045Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Persson E. Selander M. Linse S. Drakenberg T. Ohlin A.K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Abstract Full Text PDF PubMed Google Scholar, 15Rezaie A.R. Esmon C.T. J. Biol. Chem. 1994; 269: 21495-21499Abstract Full Text PDF PubMed Google Scholar, 16Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar) and a lower affinity Ca2+ binding site (kd ∼ 0.7–1.2 m M) on the isolated first EGF-like module (4Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar, 12Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (246) Google Scholar, 16Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar, 17Persson E. Hogg P.J. Stenflo J. J. Biol. Chem. 1993; 268: 22531-22539Abstract Full Text PDF PubMed Google Scholar). A Ca2+-dependent interaction between the EGF-like and Gla modules appears to enhance the affinity of the site on the EGF-like module to the point that it is tighter (17Persson E. Hogg P.J. Stenflo J. J. Biol. Chem. 1993; 268: 22531-22539Abstract Full Text PDF PubMed Google Scholar, 18Valcarce C. Holmgren A. Stenflo J. J. Biol. Chem. 1994; 269: 26011-26016Abstract Full Text PDF PubMed Google Scholar) (kd ∼ 120 μm) than the catalytic domain site. Consistent with this, nuclear magnetic resonance shows that Ca2+ binding tightens the fold of the isolated EGFN domain and bends Gla and EGFNdomains toward each other around a hinge located in the Gla domain, referred to as a helical or hydrophobic stack(19Sunnerhagen M. Olah G.A. Stenflo J. Forsen S. Drakenberg T. Trewhella J. Biochemistry. 1996; 35: 11547-11559Crossref PubMed Scopus (81) Google Scholar). Despite considerable information about Ca2+ binding to factor Xa, we have virtually no information about the location of PS binding sites on this key enzyme. The aims of this work have been to locate the lipid regulatory site(s) in factor Xa and to identify the structural domains of factor Xa necessary to see the C6PS regulatory effect on factor Xa activity. 1,2-Dicaproyl-sn-glycero-3-phospho-l-serine (C6PS) and 1,2-dicaproyl-sn-glycero-3-phosphocholine (C6PC) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Russel's viper venom factor X- activating protein (RVV-X) was purchased from Hematological Technologies Inc. (Essex Junction, VT), and factor Xa-specific substrateN-α-benzyloxycarbonyl-d-arginyl-l-glycyl-l-arginine-p-nitroanilide dihydrochloride (S-2765) was purchased from Helena Laboratories (Beaumont, TX). Chymotrypsin was purchased from Worthington (Lakewood, NJ). Chromogenetic substrate Spectrozyme PCa (SpPCa) was purchased from American Diagnostica (Greenwich, CT). [5-(Dimethylamino)-1-napthalenesulfonyl] glutamylycylarginyl chloromethyl ketone (DEGR-CK) was purchased from Calbiochem (La Jolla, CA). Diisopropyl fluorophosphate was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were ACS reagent grade or the best available grade. Bovine factor X was isolated from a barium citrate precipitate obtained from freshly collected bovine plasma (20Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (179) Google Scholar, 21Tendian S.W. Lentz B.R. Biochemistry. 1990; 29: 6720-6729Crossref PubMed Scopus (43) Google Scholar). Human factor X for stoichiometry and CD measurements was purified from recovered human plasma obtained from the American Red Cross, according to the method of Dahlback et al. (22Dahlback B. Stenflo J. Eur. J. Biochem. 1980; 104: 549-557Crossref PubMed Scopus (17) Google Scholar). Factor X obtained as above was analyzed by SDS-PAGE, concentrated (Centricon-10 concentrator supplier), and then stored at −70 °C at a concentration of about 1 mg/ml in 5 mmTris, 20 mm sodium citrate, 0.6 m NaCl, pH 7.4. A final purification of factor X was performed 1 day before an experiment by high-performance liquid chromatography on a PerkinElmer Life Sciences Isopure LC system using a Mono Q HR 5/5 ion exchange column (Amersham Biosciences, Inc., Norwalk, CN). The purified factor X was dialyzed into buffer (50 mm Tris, 175 mmNaCl, pH 7.4) for activation. Factor X (10 μm) with 5 mm Ca2+ was activated at 25 °C with RVV-X that had been covalently linked to agarose beads (10Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Crossref PubMed Scopus (75) Google Scholar, 23Nossel H. Thromb. Diath. Haemorrh. 1964; 12: 505-518PubMed Google Scholar). Factor Xa was purified by high-performance liquid chromatography on a Mono Q column, and the isolated protein was analyzed by SDS-PAGE electrophoresis. Factor Xa concentration was measured by determining the rate of S-2765 hydrolysis in a plate reader-based assay (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar), using active site-titrated factor Xa to construct the standard curve (24Jameson G.W. Roberts D.V. Adams R.W. Kyle W.S. Elmore D.T. Biochem. J. 1973; 131: 107-117Crossref PubMed Scopus (287) Google Scholar). Isolation and purification of fragments of bovine factor Xa and several of its structural domains (Gla, EGFN, Gla-EGFN, and Gla-EGFNC) by controlled trypsin digestion were described previously (18Valcarce C. Holmgren A. Stenflo J. J. Biol. Chem. 1994; 269: 26011-26016Abstract Full Text PDF PubMed Google Scholar, 25Persson E. Valcarce C. Stenflo J. J. Biol. Chem. 1991; 266: 2453-2458Abstract Full Text PDF PubMed Google Scholar,26Valcarce C. Persson E. Astermark J. Ohlin A.K. Stenflo J. Methods Enzymol. 1993; 222: 416-435Crossref PubMed Scopus (5) Google Scholar). The RSV-PL4 expression vector was used to express factor X, GDFX, and a construct missing both the Gla and first EGF domain (E2FX) in human 293 cells (16Rezaie A.R. Neuenschwander P.F. Morrissey J.H. Esmon C.T. J. Biol. Chem. 1993; 268: 8176-8180Abstract Full Text PDF PubMed Google Scholar). GDFX was also prepared for stoichiometry measurements as described by Morita and Jackson (27Morita T. Jackson C.M. J. Biol. Chem. 1986; 261: 4015-4023Abstract Full Text PDF PubMed Google Scholar) from isolated factor X. Purified factor X was reacted with α-chymotrypsin (1:400 factor X:α-chymotrypsin) at 22 °C for 45 min, a time sufficient to convert 95% of factor X to GDFX, as judged by SDS-PAGE on a 6% gel. The reaction was stopped by addition of 1 mmdiisopropyl fluorophosphate, and Gla-domainless factor X was chromatographed on a Mono Q column. GDFX Y99T mutant was also prepared as described (28Vindigni A. Winfield M. Ayala Y.M. Di Cera E. Protein Sci. 2000; 9: 619-622Crossref PubMed Scopus (7) Google Scholar, 29Rezaie A.R. J. Biol. Chem. 1996; 271: 23807-23814Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 30Guinto E.R. Vindigni A. Ayala Y.M. Dang Q.D. Di Cera E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11185-11189Crossref PubMed Scopus (47) Google Scholar). GDFX and its mutant and E2FX were activated with RVV-X as described earlier (29Rezaie A.R. J. Biol. Chem. 1996; 271: 23807-23814Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The amidolytic activities of expression products of a human factor Xa cDNA and of expression products of two of its deletion mutants (GDFXa, E2FXa) were measured in the presence of 3 mm Ca2+ using the synthetic substrate S-2765 and the microplate reader-based assay described above. Samples, containing 20 nm protein, various concentrations of C6PS and 3 mm Ca2+ in a buffer (50 mmTris, 175 mm NaCl, pH 7.6) containing 0.6% PEG, were incubated at 37 °C for 15 min before measuring activities. The amidolytic activities were estimated from measured initial rates of S-2765 hydrolysis, using a standard curve obtained with active site-titrated factor Xa (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). Amidolytic activities of GDFXa and Y99T were measured using the chromogenetic substrate Spectrozyme PCa (SpPCa) also as described earlier (29Rezaie A.R. J. Biol. Chem. 1996; 271: 23807-23814Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Samples containing 20 nm protein, various concentrations of C6PS (0, 400, 900 μm), and 0.6% PEG (to prevent adsorption of protein to the plate) were incubated in buffer (50 mm Tris, 175 mm NaCl, pH 7.6) in polypropylene Eppendorf tubes at 37 °C for 5 min before being added to a flat-bottomed polypropylene 96-well plate (Greiner America, Inc.) preincubated at 37 °C. The initial rates of SpPCa amidolysis were determined on a Versamax Tunable Microplate Reader (Molecular Devices, Sunnyvale, CA) at five substrate concentrations (50, 100, 200, 400, and 600 μm) and analyzed in terms of the Michaelis-Menten model using non-linear regression methods available in Sigma Plot 6.0. Circular dichroism (CD) spectra were generally recorded from 250 to 200 nm on an Aviv Model 620S spectrometer (Aviv Associates, Inc., Lake Wood, NJ) in a 1-cm path-length cell at 24 °C with a bandwidth of 1.0 nm. Data points were collected at every 0.5 nm with an average time of 5 s on each point. Some data were obtained down to 195 nm on an Applied Photophysics Pi* spectrometer in a 1-mm path-length cell with a bandwidth of 1 nm and data collection at every 0.5 nm. Baseline CD spectra of buffer containing various concentrations of soluble C6PS were collected in the absence and in the presence of 3 mmCa2+ and were subtracted from sample spectra. The baseline-corrected digital data were processed, smoothed, and converted to molar ellipticity, ΔΘ. We have previously determined the critical micelle concentration (CMC) of C6PS at different Ca2+ and protein concentrations (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar), but controls to detect micelle formation were in all cases still performed by watching for sudden drops in ellipticity in the range of 240–250 nm. For human and bovine factor Xa, 2J. Wang, R. Majumder, and B. R. Lentz, submitted for publication. the CMC seen in this way was similar to the CMC reported earlier by quasi-elastic light scattering methods (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). The ellipticity ratio Θ222/Θ208 (32Greenfield N.J. Anal. Biochem. 1996; 235: 1-10Crossref PubMed Scopus (564) Google Scholar) is used here as a convenient parameter to follow changes in the secondary structure of factor Xa and its fragments upon addition of C6PS. In addition, we have estimated α-helix content using published software packages CDSSTR and CONTIN (33Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2518) Google Scholar) to give context to the Θ222/Θ208 ratio. The ability of CD spectra taken to 200 nm to define α-helix content but not β-sheet or turn content is well documented (34Johnson Jr., W.C. Proteins. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar). We could not collect spectra to 185 nm to perform a complete secondary structure analysis in a buffer containing NaCl, because Na+ increases the buffer absorbance in the deep UV (34Johnson Jr., W.C. Proteins. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar). Na+ was necessary in our studies, because Ca2+ binding is required for regulation of factor Xa by C6PS (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar), and Ca2+ binding is linked to Na+ binding (35Underwood M.C. Zhong D. Mathur A. Heyduk T. Bajaj S.P. J. Biol. Chem. 2000; 275: 36876-36884Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 36Rezaie A.R. He X. Biochemistry. 2000; 39: 1817-1825Crossref PubMed Scopus (64) Google Scholar). Fluorescence intensity measurements were carried out on an SLM 48000 spectrofluorometer (SLM Aminco, Urbana, IL). Slits were closed between measurements to avoid photodegradation of the sample. All buffer solutions were filtered using 0.2-μm filters (Nalge Co., Rochester, NY). DEGR-E2FXa was prepared by sequential addition of 5 μl of DEGR-CK (1 mg/ml in 0.02 mTris, 0.1 m NaCl, pH 7.5) to 1 ml of about 1 μm purified factor E2FXa. The extent of labeling at the active site was followed by the loss of enzymatic activity, as monitored by the S-2765 assay. Labeling was stopped when no activity remained. DEGR-E2FXa was then dialyzed against 50 mm Tris, 0.1 m NaCl, pH 7.5, to remove free reagent (37Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar). DEGR-E2FXa(100 nm) in 1.0 ml of buffer (50 mm Tris, pH 7.5) was incubated in a stirred micro-cuvette (Hellma Cells, Jamaica, NY) with 0.15 mm NaCl or 3 mmCa2+ or both at 25 °C for 20 min. Following additions of C6PS (1–2 μl each addition for a maximum of 4% dilution) and an equilibration of at least 4 min, fluorescence intensity was recorded using an excitation wavelength of 340 nm (bandpass 8 nm) and an emission wavelength of 550 nm (bandpass 4 nm). For each addition, several intensity measurements were performed and averaged and corrected for dilution. Control experiments were performed in which buffer was titrated with soluble lipid in the absence of protein. The lipid solution showed very minor background fluorescence or light scattering signal (which was subtracted from sample signal) until the critical micelle concentration was reached. The critical micelle concentration for C6PS in the presence and absence of 1 μm factor Xa were determined previously to be 0.95 and 2.5 mm, respectively (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). The critical micelle concentrations for C6PC under similar conditions were even higher (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). Data were not analyzed above the critical micelle concentration. Gla-EGFNC (100 nm) in 50 mm Tris, 150 mm NaCl, pH 7.4, in the presence and in absence of 3 mm Ca2+ was titrated with soluble C6PS, and the intrinsic fluorescence was monitored at 345 nm (bandpass 4 nm) followed by excitation at 285 nm (bandpass 8 nm). Control experiments were as mentioned for DEGR-E2FXa fluorescence. C6PS and C6PC solutions were prepared from measured quantities of 10 mg/ml stock solutions in chloroform. The chloroform was evaporated under a stream of nitrogen. The lipid was re-dissolved in cyclohexane, and this solution was frozen on the wall of a capped test tube and then lyophilized overnight. The resulting dry powder was dispersed in the appropriate volume of buffer and vortexed thoroughly to reach a concentration of ∼100 mm. The final concentration of this phospholipid stock solution was determined by an inorganic phosphate assay (38Chen Jr., P.S. Toribara T.Y. Warner H. Analyt. Chem. 1956; 28: 1756-1758Crossref Scopus (5835) Google Scholar). The stoichiometries of soluble C6PS binding to factor Xa, GDFXa, Gla-EGFNC, and Gla in the absence and in the presence of 3 mm Ca2+ were determined by equilibrium dialysis measurements. This procedure not only establishes stoichiometry but also confirms indirect binding results by a direct measurement. Experiments were performed using 2.0-ml Teflon dialysis cells (Spectrum Medical, Los Angeles, CA) with the two cells separated by a 2000 molecular weight cut-off membrane. Both chambers contained equal amounts of C6PS, enough to saturate >85% of the protein present in one-half of the dialysis cell at varying concentrations (30–100 μm). The lipid concentrations used depended on the crude binding constants estimated in CD titrations. Depending on the particular combination of lipid and protein concentration in a given experiment and on the stoichiometry of binding for a particular peptide, between 79 and 94% of lipid remained unbound at equilibrium. The two chambers were allowed to equilibrate at room temperature for 24 h while being rotated horizontally at 20 rpm. The protein concentration gradient between the two halves of the cell causes a difference in the total phosphate concentration between the two halves of the cell. The concentration of protein-bound C6PS was measured as the difference in total phosphate concentration (ΔP) (38Chen Jr., P.S. Toribara T.Y. Warner H. Analyt. Chem. 1956; 28: 1756-1758Crossref Scopus (5835) Google Scholar) between the two chambers of the dialysis cell. Assuming a simple model of binding of lipid to n equivalent and independent sites, it is easy to show that ΔP should vary with protein concentration as follows, ΔP=[L]n[P]kdn+[L]Equation 1 where [L] is free lipid concentration andkd/n is the observed stoichiometric dissociation constant for lipid binding, assuming a single site model. For [L] ≫ kd/n, this is roughly a straight line with a slope proportional to n. In our experiments, we maintained [L] >kd/n, but the total lipid concentration had to remain less than the CMC of the lipid. Thus, to obtainn, we had to fit a plot of ΔP versusprotein concentration to the non-linear equation given in Equation 1, using standard non-linear regression procedures and the program SigmaPlot (version 6 for Windows 2000; Jandel Scientific). In our experiments, soluble lipid was added to the protein solution, and the observed response was taken as representing the fraction of protein bound (f) to lipid at concentration [L] given by,f=[L]Kd+[L]Equation 2 where Kd is the apparent stoichiometric binding constant for soluble lipid binding to the protein. Any observable value that changes from an initial value ofR0 to a final value at saturation,Rsat, as a result of binding can be written as follows,RR0=1+Rsat−R0R0×fEquation 3 We have shown previously that C6PS enhances proteolytic activity of human factor Xa by roughly 60-fold but inhibits the amidolytic activity toward S-2765 by 60% (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). The variation of amidolytic activity of expressed human factor Xa, GDFXa, and E2FXa (Fig. 1) in the presence of 3 mm Ca2+ with soluble C6PS concentration is shown in Fig. 2. The highest C6PS concentration used (0.8 mm) was still below the CMC for C6PS in the presence of 3 mm Ca2+(∼2.5 mm) (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). The amidolytic activities of factor Xa and GDFXa were decreased by 79 and 9%, respectively, at saturation with C6PS (TableI), but C6PS had negligible effect on the amidolytic activity of E2FXa. The functional responses of factor Xa and its constructs to C6PS binding were reasonably well described by a single binding site model (see “Methods”), and the binding parameters are given in Table I. The apparent Kd for C6PS binding to the expressed human factor Xa in this experiment (39 ± 6 μm) was comparable to but somewhat smaller than we have reported previously for factor Xa isolated from outdated human plasma (65 ± 5 μm) (1Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar), and the percent inhibition was also greater (80 versus 60%). This probably reflects the slight difference between Xa from human plasma and factor Xa from a single cDNA clone, as used here.Table IParameters describing binding of soluble C6PS to human factor Xa and its constructs in terms of changes in amidolytic activityProtein/domainKd1-aKd, apparent stoichiometric dissociation constant.ΔRsat1-bΔRsat, percent change in activity upon addition of saturating concentration of C6PS.μmHuman Xa39 ± 6−79Human GDFXa86 ± 18−9Human E2FXaNA1-cNA, not applicable.01-a Kd, apparent stoichiometric dissociation constant.1-b ΔRsat, percent change in activity upon addition of saturating concentration of C6PS.1-c NA, not applicable. Open table in a new tab The effects of soluble C6PS on the CD spectra of expressed human factor Xa and its constructs, GDFXa and E2FXa, were studied in the absence and presence of 3 mmCa2+. CD spectra of human factor Xa are shown at various concentrations of C6PS in the presence (Fig.3A) and in the absence of 3 mm Ca2+ (Fig. 3B). Although it is reported to bind Ca2+ (17Persson E. Hogg P.J. Stenflo J. J. Biol. Chem. 1993; 268: 22531-22539Abstract Full Text PDF PubMed Google Scholar), human factor Xa did not undergo a detectable change in secondary structure upon addition of 3 mm Ca2+, as seen from the solidand dotted curves in Fig. 3A and from the Θ222/Θ208 ratio and α-helical content (Table I). Secondary structure analysis yielded an estimate of 11% helical content in the presence or absence of Ca2+, in good agreement with the reported helicity for the analogous factor IXa crystal structure (10.6%) (8Brandste" @default.
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• W2028776941 title "Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa" @default.
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