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1.
The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin 总被引:15,自引:0,他引:15
R E Jordan G M Oosta W T Gardner R D Rosenberg 《The Journal of biological chemistry》1980,255(21):10081-10090
The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide. 相似文献
2.
Michael W. Piepkorn David Lagunoff Gottfried Schmer 《Biochemical and biophysical research communications》1978,85(2):851-856
Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 106 M?1 for the low molecular weight preparation and 2.89 × 106 M?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity. 相似文献
3.
Michael W. Piepkorn David Lagunoff Gottfried Schmer 《Archives of biochemistry and biophysics》1980,205(2):315-322
Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (Ka) of 4.9 × 105, m?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent Ka = 7.9 × 105, m?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately. 相似文献
4.
M J Griffith H S Kingdon R L Lundblad 《Biochemical and biophysical research communications》1979,87(3):686-692
Active site blocked-thrombin, prepared by reacting thrombin with valyl-isoleucyl-prolyl-arginine chloromethyl ketone, inhibits the heparin enhanced-antithrombin III/thrombin reaction. Since active site blocked-thrombin does not interact with antithrombin III it was concluded that active site blocked-thrombin was competing for heparin in the reaction system. The heparin concentration dependence for maximum enhancement of the antithrombin III/thrombin reaction in the presence and absence of active site blocked-thrombin indicated that heparin was binding to thrombin to enhance the reaction rate. A dissociation constant value of 6.4×10?9M was estimated for the heparin·thrombin complex which is similar to the value of 5.8×10?9M previously reported (Griffith M.J. (1979) in press). Antithrombin III·thrombin complexes were also found to bind heparin with an affinity equivalent to thrombin. The results were interpreted to indicate that heparin binds to thrombin as the first step in the mechanism of action of heparin in enhancing the antithrombin III/thrombin reaction. 相似文献
5.
R E Jordan G M Oosta W T Gardner R D Rosenberg 《The Journal of biological chemistry》1980,255(21):10073-10080
A low molecular weight preparation of porcine heparin (specific anticoagulation activity = 125 units/mg) was fractionated to obtain a mucopolysaccharide product of 6500 daltons (specific anticoagulant activity = 373 units/mg) that is homogeneous with respect to its interaction with antithrombin. This material was treated with fluorescamine in order to introduce a fluorescent tag into the mucopolysaccharide. Initially, we showed that the fluorescamine-heparin conjugate and the unlabeled mucopolysaccharide interacted with antithrombin in a virtually identical fashion. Subsequently, we demonstrated that labeled heparin could be utilized in conjunction with fluorescence polarization spectroscopy to monitor the binding of mucopolysaccharide to thrombin, factor IXa, factor Xa, and plasmin. The interaction of this complex carbohydrate with thrombin exhibited a stoichiometry of 2:1 with KH1T DISS = KH2T DISS = 8 x 10(-7) M. The formation of mucopolysaccharide . factor IXa complex is characterized by a stoichiometry of 1:1 with KHIXa DISS = 2.58 x 10(-7) M. The binding of heparin to factor Xa or plasmin occurred with low avidity. Therefore, the stoichiometries of these processes could not be established. However, our experimental data were compatible with a single-site binding residue with KHXa DISS = 8.73 x 10(-6) M and KHPL DISS = approximately 1 x 10(-4) M, respectively. 相似文献
6.
The association of heparin with thrombin was investigated by fluorometric titration. A maximum of 25% of the fluorescence of fluorescein-labeled heparin (FTC-heparin) was quenched at thrombin saturation in the absence of NaCl. FTC-heparin (H) associated tightly with thrombin (T) and the association constant of the ternary complex, H2T, formed in the absence of NaCl, was calculated to be 1.7 × 108M?1. However, the association was strongly influenced by the NaCl concentration, and the association constant of the equimolar complex, HT, formed in 0.15M NaCl was found to be 1 × 106M?1. The first-order rate constant, kapp, for inactivation of thrombin by antithrombin III (AT III) and low-affinity heparin (LA-heparin) was comparable with that of high-affinity heparin (HA-heparin) in the absence of NaCl, but decreased with an increase in the concentration of NaCl. The decreased enhancement of the thrombin-AT III reaction by LA-heparin at high NaCl concentration appeared to result from a decreased association of thrombin with LA-heparin, thus reducing the formation of the ternary complex, thrombin-LA-heparin-AT III. 相似文献
7.
The inhibition of activated bovine coagulation factors X and VII by antithrombin III 总被引:1,自引:0,他引:1
J Jesty 《Archives of biochemistry and biophysics》1978,185(1):165-173
The inhibition of activated bovine Factors VII and X by antithrombin III has been studied by kinetic methods. The reaction between Factor Xa and antithrombin III is characterized by second-order kinetics, with a rate constant of 3.9 × 103m?1s?1 at pH 7.5 at 37 °C. Inhibition in the presence of excess antithrombin III does not proceed to completion: The decay of Factor Xa deviates from pseudo-first-order kinetics and a final equilibrium is reached, suggesting reversibility. The apparent association constant, at pH 7.5, 37 °C, is 2.3 × 109m?1. The interaction of three forms of bovine Factor VII with antithrombin III has been studied by the same methods. Factor VII and the two-chain activated form, α-Factor VIIa, and the tissue factor-Factor VIIa complex are not significantly inhibited by plasma levels of antithrombin III, in the either the presence or absence of heparin. 相似文献
8.
Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength (K0), and molar extinction difference coefficients (Δ?λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K0 = 1.3 × 10?3m and Δ?480 = 1.5 × 104m?1 cm?1; for ArsI, K0 = 5.8 × 10?3m and Δ?562 = 1.8 × 104m?1 cm?1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms. 相似文献
9.
Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors 总被引:3,自引:0,他引:3
Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I. 相似文献
10.
C H Pletcher M T Cunningham G L Nelsestuen 《The Journal of biological chemistry》1986,261(9):4143-4147
The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function. 相似文献
11.
Somatostatin binding to pituitary plasma membranes. 总被引:3,自引:0,他引:3
J W Leitner R M Rifkin A Maman K E Sussman 《Biochemical and biophysical research communications》1979,87(3):919-927
A method has been developed for the study of somatostatin binding to anterior pituitary plasma membranes. When 5×10?9M [125I]Tyr1-somatostatin (SA 18 Ci/mmol) was incubated with isolated pituitary plasma membranes (protein = 100 μg), 13.6% of total radioactivity was bound excluding nonspecific binding. The Scatchard plot could be resolved into two distinct components and analyzed to yield: K1diss = 3.3×10?8M and K2diss = 7.7×10?6M. This binding was shown to be specific for somatostatin. 相似文献
12.
Byung K. Kim Manfred Steiner Mario G. Baldini 《Biochemical and biophysical research communications》1980,97(3):1227-1232
The receptor site for antithrombin III (AT III) was investigated in normal human platelets. [125I] iodinated AT III was utilized as tracer for the binding assay. Equilibrium of AT III binding was reached within 2 min. The binding capacity was pH-dependent with the optimum around pH 7.0. Binding specificity was demonstrated by inhibition of [125I] AT III ligation using an excess amount of non-labeled AT III. The AT III·heparin complex did not supress [125I] AT III binding. Analysis of binding data by Scatchard plot revealed a single class of binding sites with Kd of 3.2 × 10?7 M and binding capacity of 3840 per platelet. 相似文献
13.
James C. Fredenburgh Beverly A. Leslie Alan R. Stafford Teresa Lim Howard H. Chan Jeffrey I. Weitz 《The Journal of biological chemistry》2013,288(41):29394-29402
The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn2+ in this interaction because Zn2+ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn2+ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn2+-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn2+-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn2+ is present. These results reveal the mechanism by which Zn2+ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin. 相似文献
14.
Kinetic properties of rat hepatic prolactin receptors 总被引:1,自引:0,他引:1
Binding of 125I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent Kd of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K1, of 2.1 × 107, 1.4 × 107, 1.2 × 107 and 4 × 106 M?1. min?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K?1=1.26 × 10?2. min?1 () and a slower phase with K?1=1.103 × 10?3. min?1. The apparent Kd (from K?1K1) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone. 相似文献
15.
Significant differences between saturation kinetic properties of heparin-stimulated reactions between thrombin and antithrombin III from human and bovine species were observed. In both systems, the apparent Km for antithrombin III was higher than the KD for antithrombin III-heparin interaction, monitored by intrinsic protein fluorescence change. The Km for thrombin and kcat were much higher for proteins of the human species than the bovine species. The apparent Km for one human protein was dependent on the concentration of the other human protein, indicating interaction of the binding events. The reaction product formed from the bovine proteins was a potent inhibitor of the reaction but the product from the human proteins was a poor inhibitor. The major differences between the two species appeared to be related to interaction of thrombin or thrombin derivatives with heparin or heparin-antithrombin III complexes. 相似文献
16.
I. N. Sokotun O. V. Gnedenko A. V. Leychenko M. M. Monastyrnaya E. P. Kozlovskaya A. A. Molnar A. S. Ivanov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(2):139-142
The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: KD = 7.38 × 10?8 M and 9.93 × 10?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively. 相似文献
17.
L-T3 transport has been investigated in human red cell ghosts. Determination of initial T3 uptake revealed two separate saturable uptake systems, one with a Km of 1.6 × 10?8M, the other with a Km of 3.3 × 10?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10?7M.and 2.6 × 10?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T3. The T3 was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low Km system, but was without effect on the high Km system. Thus evidence is provided both of binding of T3 to the ghost membrane and of its uphill transport across the membrane. 相似文献
18.
A colorimetric receptor L was prepared. Receptor L can selectively sense F? based on distinct color changes among a series of ions. It can selectively sense F? through an intramolecular hydrogen bond interaction. A Job plot indicated a 1:1 complexation stoichiometry between receptor L and F?. The association constant for L –F? in CH3CN was determined as 9.70 × 104 M?1 using a Stern–Volmer plot. 相似文献
19.
The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH3)4NCl) or choline chloride (HO(CH2)2N(CH3)3Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na+ enhanced thrombin activity by decreasing the Km,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average KA for Na+for the four substrates examined was 3.5 × 10?2m. A comparison of the effects of Na+ vs K+ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the Km,app (0.2 to 04.-fold) and increased thekcat,app (3.1 to 3.4-fold) except that higher K+ concentrations (KA = 2.8 × 10?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na+ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?288.5 = +1067). The Km for Na+ was 2.3 × 10?2m which agrees with the kinetically determined KA for Na+. The results are consistent with Na+ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme. 相似文献
20.
《International journal of biological macromolecules》1987,9(4):225-232
The highest antithrombogenic activity was achieved by the sulphation of partially N-deacetylated O-(carboxymethyl)chitin among variously modified chitin derivatives. It was also suggested that the distribution of N-sulphate and N-acetyl groups on the C-2 position might be essential to the selective binding by antithrombin-III to inhibit thrombin activity. Kinetic evaluations demonstrated the non-competitive inhibition on direct interaction with thrombin (Ki = 9.26 × 10−8m) and the competitive inhibition with antithrombin-III (Ki = 3.33 × 10−8) m as well as with heparin. 6-O-Carboxymethyl groups were found, from the data of intravenous injection in mice, to suppress the toxicity of chitin heparinoids. 相似文献