IS PENTASACCHARIDE THE SMALLEST HEPARIN OLIGOSACCHARIDE WITH BINDING AFFINITY TO ANTITHROMBIN III ? AN OVERVIEW

1987 ◽  
Author(s):  
J Mardiguian

The binding of heparin to antithrombin III is ascribed to the presence in the heparin molecule of a specific binding site which contains a typical 3-0- sulfate group located on a glucosamine residue. It has been postulated that the smallest heparin oligosaccharide capable of high affinity binding to antithrombin III and eliciting anti-factor Xa activity is a pentasaccharide containing three glucosamine units and two uronic acid residues. Such a pentasaccharide has been recently isolated after chemical depolymerization of pig mucosal heparin and its structure found to be very close to that of a synthetic pentasaccharide prepared by other investigators. However no convincing data have, so far, excluded the possibility that an oligosaccharide composed of less than five sugar units could not be able to bind to antithrombinlll and to elicit anti-factor Xa activity. We report now the isolation of new oligosaccharides obtained by beta-eliminative chemical depolymerization of heparin using three different procedures : depolymerization of heparin benzyl ester (1) in aqueous medium and (2) in non-aqueous medium: (3) alcaline depolymerization of a periodate oxydized acetyl heparin. The data reported show that the high affinity oligosaccharides isolated after affinity chromatography on immobilized antithrombin III are distinct from the previously isolated pentasaccharide and that there is some evidence that these are tetrasaccharides resulting from the cleavage of the non reducing end of the heparin molecule

1981 ◽  
Vol 197 (3) ◽  
pp. 599-609 ◽  
Author(s):  
B Casu ◽  
P Oreste ◽  
G Torri ◽  
G Zoppetti ◽  
J Choay ◽  
...  

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].


Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372 ◽  
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.


1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.


2003 ◽  
Vol 46 (24) ◽  
pp. 5184-5195 ◽  
Author(s):  
Gudrun Lange ◽  
Dominique Lesuisse ◽  
Pierre Deprez ◽  
Bernard Schoot ◽  
Petra Loenze ◽  
...  

1991 ◽  
Vol 11 (4) ◽  
pp. 1777-1784 ◽  
Author(s):  
Y D Halvorsen ◽  
K Nandabalan ◽  
R C Dickson

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.


Biochemistry ◽  
1998 ◽  
Vol 37 (10) ◽  
pp. 3321-3328 ◽  
Author(s):  
George M. Willems ◽  
Marie P. Janssen ◽  
Irene Salemink ◽  
Tze-Chein Wun ◽  
Theo Lindhout

1982 ◽  
Vol 37 (7-8) ◽  
pp. 620-631 ◽  
Author(s):  
Henrik Laasch ◽  
Klaus Pfister ◽  
Wolfgang Urbach

Abstract High- and low-affinity binding of photosystem II herbicides to isolated thylakoids of Spinacia oleracea and to intact cells of the unicellular green alga Ankistrodesmus braunii were investigated. Complete mutual displacement of bound diuron-type herbicides (e.g. diuron, atrazine, terbutryn) by either diuron- or phenol-type herbicides (e.g. ioxynil, dinoseb) in thylakoids as well as in intact algal cells was found for herbicide concentrations (< 4 nmol bound herbicide/mg Chl) which gave almost saturated high-affinity binding. This demonstrates a high degree of specific binding of these herbicides towards their receptor sites even in intact algal cells. In contrast, phenol-type herbicides are largely unspecifically bound in algal cells. The mechanism of binding of all photosystem II herbicides at the high-affinity (specific) binding site was found to be competitive. Within the group of diuron-type and of phenol-type herbicides as well as between these two groups, graphical and quantitative analysis of the Lineweaver- Burk plot and of the Dixon plot indicated competitive binding. From this a common binding site for both types of herbicides was concluded. The involvement of two different herbicide binding- proteins is discussed. Low-affinity (unspecific) binding was found to be irreversible in contrast to the easily reversible high-affinity binding. Irreversibility was indicated by a lack of displacement. It is proposed that low-affinity binding represents either a partitioning of the herbicides into the lipophilic parts of the membranes or an attachment to distinct receptor sites. Unspecifically bound herbicides might be responsible for several high concentration effects of the photosystem II herbicides, which are described in the literature. Evidences for the possible existence of a second binding site of these herbicides are presented.


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