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Activation of heparin cofactor II by heparin oligosaccharides 总被引:1,自引:0,他引:1
M M Maimone D M Tollefsen 《Biochemical and biophysical research communications》1988,152(3):1056-1061
Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required. 相似文献
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Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments 总被引:1,自引:0,他引:1
P Schoen T Lindhout G Willems H C Hemker 《The Journal of biological chemistry》1989,264(17):10002-10007
Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin. 相似文献
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Antithrombin, the main inhibitor of thrombosis in blood, is bound and activated by the heparin-like side-chains that line the small vasculature. We now have good depictions of the heparin-binding site on antithrombin, and of the way in which mutations at this site cause thrombotic disease. The interaction of heparin with antithrombin is, however, a kinetic one, with binding being followed by formation of a complex with thrombin and then release from the heparin. Our understanding of the processes involved is currently based on crystallographic models but, for a mobile mechanism, these merely provide snapshots - what is needed is a movie. 相似文献
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Johan Riesenfeld Inger Pettersson Ulf Lindahl Willie F Vann Lennart Roden 《Glycoconjugate journal》1987,4(2):179-189
The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.Abbreviations PAPS
3-phosphoadenylylsulfate
- Hepes
4-(2-hydroxy-ethyl)piperazineethanesulfonic acid
- GlcUA
glucuronic acid
This is Paper XIV of a series in which the preceeding reports are refs 10 and 12. A preliminary report has appeared [28]. 相似文献
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Laercio Pol‐Fachin Camila Franco Becker Jorge Almeida Guimarães Hugo Verli 《Proteins》2011,79(9):2735-2745
Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β‐AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135‐linked oligosaccharide caused a bending in AT‐bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α‐AT. The obtained data supported the proposal of an atomic‐level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β‐sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135‐linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic‐level explanation for the Asn135‐linked glycan influence over heparin binding and AT activation. Proteins 2011; © 2011 Wiley‐Liss, Inc. 相似文献
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Role of lysine 173 in heparin binding to heparin cofactor II 总被引:1,自引:0,他引:1
H C Whinna M A Blinder M Szewczyk D M Tollefsen F C Church 《The Journal of biological chemistry》1991,266(13):8129-8135
Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC. 相似文献
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The effects of bivalent cations on heparin binding, structure, and thrombin inhibition rates of heparin cofactor II were examined. Zn(2+) - and to a lesser extent Cu(2+) and Ni(2+) - enhanced the interaction between heparin cofactor II and heparin as demonstrated by heparin affinity chromatography and surface plasmon resonance experiments. Metal chelate chromatography and increased intrinsic protein fluorescence in the presence of Zn(2+) indicated that heparin cofactor II has metal ion-binding properties. The results are compatible with the hypothesis that Zn(2+) induces a conformational change in heparin cofactor II that favors its interaction with heparin. 相似文献
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The properties of the metachromatic dye toluidine blue have been utilized to determine colorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations. 相似文献
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M Fath V VanderNoot I Kilpel?inen T Kinnunen H Rauvala R J Linhardt 《FEBS letters》1999,454(1-2):105-108
The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction. 相似文献
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