scholarly journals Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide

1995 ◽  
Vol 309 (2) ◽  
pp. 465-472 ◽  
Author(s):  
N Razi ◽  
E Feyzi ◽  
I Björk ◽  
A Naggi ◽  
B Casu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Binding Sites ◽  
Capsular Polysaccharide ◽  
Factor Xa ◽  
Compositional Analysis ◽  
Basic Structure ◽  
Sulphate Content ◽  
High Affinity ◽  
Structural And Functional Properties

Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res. 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT). A high-affinity subfraction, M(r) approx. 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx. 1 microM, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.

scholarly journals Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions

1991 ◽  
Vol 275 (1) ◽  
pp. 151-158 ◽  
Author(s):  
M Kusche ◽  
H H Hannesson ◽  
U Lindahl
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Binding Sites ◽  
Proteinase Inhibitor ◽  
Capsular Polysaccharide ◽  
Polymer Modification ◽  
Sulphated Polysaccharide ◽  
E Coli ◽  
High Affinity ◽  
K5 Polysaccharide

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)→(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3′-phosphate 5′-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

1979 ◽  
Author(s):  
B. Kudryk ◽  
M. Blombäck
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Specific Activity ◽  
High Specific Activity ◽  
Antigenic Determinants ◽  
Affinity Binding ◽  
Compact Structure ◽  
High Affinity Binding ◽  
High Affinity ◽  
Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system

2019 ◽  
Vol 2 (3) ◽  
pp. e201900368 ◽  
Author(s):  
Yasuhiro Yunoki ◽  
Kentaro Ishii ◽  
Maho Yagi-Utsumi ◽  
Reiko Murakami ◽  
Susumu Uchiyama ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Conformational Changes ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Periodic Oscillation ◽  
Mass Spectrometric ◽  
High Affinity ◽  
Clock System ◽  
Determining Factor

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB–KaiC interaction; however, its relationship with the KaiA–KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.

Substrate recognition and translocation by polyspecific organic cation transporters

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Hermann Koepsell
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Organic Cation ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Organic Cation Transporters ◽  
Cationic Drugs ◽  
High Affinity ◽  
Cation Transporters ◽  
Substrate Binding Sites

Abstract Organic cation transporters (OCTs) of the SLC22 family play a pivotal role in distribution and excretion of cationic drugs. They mediate electrogenic translocation of cations in both directions. OCTs are polyspecific transporters. During substrate translocation they perform a series of conformational changes involving an outward-facing conformation, an occluded state and an inward-facing conformation. Mutagenesis of OCT1 in combination with homology modeling showed that identical amino acids form the innermost parts of the outward-open and inward-open binding clefts. In addition to low affinity substrate binding sites, OCT1 contains high affinity substrate binding sites that can mediate inhibition via non-transported compounds.

scholarly journals High affinity cation-binding sites in Complex I from Escherichia coli

2009 ◽  
Vol 1787 (8) ◽  
pp. 1024-1028 ◽  
Author(s):  
Liliya Euro ◽  
Galina Belevich ◽  
Mårten Wikström ◽  
Marina Verkhovskaya
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Complex I ◽  
Cation Binding ◽  
High Affinity

Multimerin 1 binds factor V and activated factor V with high affinity and inhibits thrombin generation

2008 ◽  
Vol 100 (12) ◽  
pp. 1058-1067 ◽  
Author(s):  
Samira B. Jeimy ◽  
Nola Fuller ◽  
Subia Tasneem ◽  
Kenneth Segers ◽  
Alan R. Stafford ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor V ◽  
Cell Secretion ◽  
High Affinity ◽  
Secretion Granules ◽  
Functional Consequences ◽  
Membrane Binding Sites

SummaryMultimerin 1 (MMRN1) is a polymeric, factorV (FV) binding protein that is stored in platelet and endothelial cell secretion granules but is undetectable in normal plasma. In human platelet α-granules, FV is stored complexed to MMRN1, predominantly by noncovalent binding interactions. The FV binding site for MMRN1 is located in the light chain, where it overlaps the C1 and C2 domain membrane binding sites essential for activated FV (FVa) procoagulant function. Surface plasmon resonance (SPR), circular dichroism (CD) and thrombin generation assays were used to study the binding of FV and FVa to MMRN1, and the functional consequences. FV and FVa bound MMRN1 with high affinities (KD:2 and 7 nM, respectively). FV dissociated more slowly from MMRN1 than FVa in SPR experiments, and CD analyses suggested greater conformational changes in mixtures of FV and MMRN1 than in mixtures of FV and MMRN1. SPR analyses indicated that soluble phosphatidylserine (1,2-Dicaproylsn-glycero-3-phospho-L-serine) competitively inhibited both FV-MMRN1 and FVa-MMRN1 binding. Furthermore, exogenous MMRN1 delayed and reduced thrombin generation by plasma and platelets, and it reduced thrombin generation by preformed FVa. Exogenous MMRN1 also delayed FV activation, triggered by adding tissue factor to plasma, or by adding purified thrombin or factor Xa to purified FV. The high affinity binding of FV to MMRN1 may facilitate the costorage of the two proteins in platelet α-granules. As a consequence, MMRN1 release during platelet activation may limit platelet dependent thrombin generation in vivo.

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