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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.
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PMID:Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups. 141 23

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.
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PMID:Search for the heparin antithrombin III-binding site precursor. 173 39

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.
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PMID:Influence of tryptophan modification upon digestion of antithrombin III by elastase. 205 15

According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.
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PMID:Influence of chemical modification of tryptophan residues on the properties of human antithrombin III. 231 91

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.
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PMID:Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin. 241 80

Computer simulation studies were used to prepare an ensemble of heparin number chains. The polydispersity of these chains was simulated by introducing a specific "fraction of terminators", and it closely resembled the experimentally observed polydispersity of a porcine mucosal, glycosaminoglycan heparin. The same percentage of simulated chains contained antithrombin III (ATIII) binding site sequences as are typically found to contain ATIII binding sites using affinity chromatography. Heparin lyase action was then simulated by using Michaelis-Menten kinetics. In one model, heparin chains were constructed from the random assembly of monosaccharide units using the observed mole percentage of each. After simulated depolymerization, the final oligosaccharides formed were compared to the observed oligosaccharide products. The simulation which assumed a random distribution of monosaccharide units in heparin did not agree with experimental observations. In particular, no ATIII binding site sequences were found in the simulated number chains. The results of this simulation indicate that heparin is not simply a random assembly of monosaccharide units. These results are consistent with the known, ordered biosynthesis of heparin. In a second model, heparin chains were constructed from randomly assembled oligosaccharides at the mole percentage in which each is found in the final product mixture. The action of heparin lyase was then simulated, and the distribution of the oligosaccharide products was measured throughout the simulated time course of the depolymerization reaction. The simulated rate of formation and final concentration of a particular oligosaccharide which contains a portion of heparin's ATIII binding site were similar to those observed experimentally. These results are consistent with the random distribution of ATIII binding sites within glycosaminoglycan heparin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nonrandom structural features in the heparin polymer. 274 16

1. One can accurately predict the contribution of each inhibitor to the total inactivation of an enzyme in plasma once its pseudo-first-order reaction rate constant and concentration are known. 2. Because the mechanism of augmentation of the inactivation rate of an enzyme by ATIII occurs via formation of an ATIII-heparin complex, the degree of potentiation can be predicted by knowing the binding capacity (sites per mole) of the heparin preparation and the concentration of heparin in the reaction (to calculate the concentration of the ATIII-heparin complex). 3. The augmentation by heparin of the inactivation rate of a particular enzyme by ATIII is dependent upon the presence of other enzymes with higher kassoc, since these would strongly compete for the ATIII-heparin complex. 4. In a plasma environment, using therapeutic levels of heparin, there is no augmentation of the inactivation rate of any of the contact enzymes.
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PMID:Effect of heparin on the inhibition of the contact system enzymes. 278 90

Although the effect of aspirin in blood coagulation has been believed to be due to its ability to interfere with platelet function, very few studies have reported its effect on various blood coagulation proteins. Since aspirin (acetylsalicylic acid) is known to acetylate numerous biologic macromolecules, the effect of aspirin on antithrombin III was investigated. It was found that antithrombin III is irreversibly inactivated by treatment with aspirin. The inactivation follows pseudo first-order kinetics and incorporation of one molecule of aspirin per molecule of the protein is necessary for complete inactivation. Reaction with acetyl-[14C]-salicylic acid incorporated 1.4 mol of acetyl group per mole of protein but reaction with carboxyl-[14C]-acetyl salicylic acid incorporated only 0.03 mol of radioactive label per mole of the protein. Furthermore, sodium salicylate does not inactivate the protein. This suggests that the reaction occurs through the acetylation of antithrombin III. Amino group analysis of aspirin-treated antithrombin III using trinitrobenzenesulfonic acid revealed that one to two primary amino groups are lost relative to the untreated antithrombin III. It is concluded that the reaction of aspirin with antithrombin III results in specific acetylation of lysine residues.
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PMID:Acetylation of antithrombin III by aspirin. 309 26

This study characterizes the structural and functional significance of sulfhydryl residues in human plasma heparin cofactor II (HCII). For quantification of sulfhydryl groups, the extinction coefficient of HCII was redetermined and found to be 0.593 ml mg-1 cm-1 using second-derivative spectroscopy and multicomponent analysis assuming 4, 10, and 2 residues of tryptophan, tyrosine, and tyrosine-O-sulfate per mole of protein, respectively. The results show that tyrosine-O-sulfate residues in HCII and in cholecystokinin peptide fragments (as model compounds) do not significantly contribute to the absorbance spectrum from 280 to 300 nm. A total of three sulfhydryl groups per mole of HCII was detected by Ellman's reagent titration, with or without treatment with dithioerythritol, indicating the absence of intramolecular disulfide bonds. Incubation of HCII with 0.1-10 mM dithioerythritol did not diminish its heparin-enhanced thrombin inhibition activity. Treatment with various sulfhydryl-specific reagents, including p-mercuribenzoate, HgCl2, and N-substituted maleimide derivatives, inactivated HCII. Titration with Ellman's reagent after these reactions identified the modification site as a cysteinyl residue(s). However, complete methanethio derivatization of the sulfhydryl groups of HCII using methyl methanethiosulfonate did not alter heparin-catalyzed thrombin inhibition. These results indicate that the sulfhydryl groups of HCII are not essential for thrombin inhibition. HCII differs from antithrombin III, which contains an essential disulfide bond for heparin-dependent thrombin inhibition (Longas, M. O., et al. (1980) J. Biol. Chem. 255, 3436). Furthermore, within the "serpin" (serine proteinase inhibitor) superfamily, HCII resembles chicken ovalbumin in occurrence of sulfhydryl residues and reactivity with various sulfhydryl group-directed compounds.
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PMID:Structure-function relationships in heparin cofactor II: spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition. 342 30

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.
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PMID:Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange. 643 45


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