scholarly journals The molecular-weight-dependence of the anti-coagulant activity of heparin

1978 ◽  
Vol 175 (2) ◽  
pp. 691-701 ◽  
Author(s):  
T C Laurent ◽  
A Tengblad ◽  
L Thunberg ◽  
M Höök ◽  
U Lindahl
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Random Distribution ◽  
Degree Of Polymerization ◽  
Molecular Weight Range ◽  
High Affinity ◽  
Predicted Probability ◽  
Coagulant Activity ◽  
The Relationship ◽  
Molecular Weight Fractions

It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.

scholarly journals A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

1990 ◽  
Vol 1 (11) ◽  
pp. 843-852 ◽  
Author(s):  
H McNeill ◽  
P J Jensen
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Human Keratinocytes ◽  
Urokinase Plasminogen Activator ◽  
Receptor Expression ◽  
Epithelial Migration ◽  
High Affinity ◽  
Upa Receptor

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.

scholarly journals Tuning the Properties of High Molecular Weight Chitosans to Develop Full Water Solubility Within a Wide pH Range

2020 ◽  
Author(s):  
Anderson Fiamingo ◽  
Sergio Paulo Campana Filho ◽  
Osvaldo Novais Oliveira Junior
Keyword(s):  
Molecular Weight ◽  
Biomedical Applications ◽  
Random Distribution ◽  
Water Solubility ◽  
Aqueous Media ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
H Nmr ◽  
Wide Ph Range ◽  
Ph Range

<p>The preparation of chitosans soluble in physiological conditions has been sought for years, but so far solubility in non-acidic aqueous media has only been achieved at the expense of lowering chitosan molecular weight. In this work, we applied the multistep ultrasound-assisted deacetylation process (USAD process) to β-chitin and obtained extensively deacetylated chitosans with high molecular weights (Mw ≥ 1,000,000 g mol<sup>-1</sup>). The homogeneous <i>N</i>-acetylation of a chitosan sample resulting from three consecutive USAD procedures allowed us to produce chitosans with a high weight average degree of polymerization (DPw ≈ 6,000) and tunable degrees of acetylation (DA from 5 to 80%). <i>N</i>-acetylation was carried out under mild conditions to minimize depolymerization, while preserving a predominantly random distribution of 2-amino-2-deoxy-D-glucopyanose (<i>GlcN</i>) and 2-acetamido-2-deoxy-D-glucopyanose (<i>GlcNAc</i>) units. This close to random distribution, inferred with deconvolution of nuclear magnetic resonance (<sup>1</sup>H NMR) spectra, is considered as responsible for the solubility within a wide pH range. Two of the highly <i>N</i>-acetylated chitosans (DA ≈ 60 % and ≈ 70 %) exhibited full water solubility even at neutral pH, which can expand the biomedical applications of chitosans. </p>

scholarly journals Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1306-1311 ◽  
Author(s):  
SR Reddigari ◽  
P Kuna ◽  
G Miragliotta ◽  
Y Shibayama ◽  
K Nishikawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Light Chains ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells

Abstract High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. Excision of the vasoactive peptide bradykinin by plasma kallikrein results in kinin-free HK that consists of a 65-Kd N-terminal heavy chain (HK-HC) linked to the C-terminal 45- Kd light chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor of SH-proteases and HK-LC contains the binding sites for coagulation cofactors prekallikrein and factor XI. HK has previously been shown to bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc(2+)-dependent manner by a single class of high-affinity binding sites. We have further characterized that interaction in order to determine the cell-binding regions of HK. Competition binding experiments have indicated that either HK-LC or HK-HC was able to inhibit the binding of labeled HK with a 50% inhibitory concentration (IC50) of 77 nmol/L and 89 nmol/L, respectively. Cleaved two-chain HK (HKa) had an IC50 of 73 nmol/L, whereas uncleaved HK had an IC50 of 335 nmol/L. Direct binding experiments have indicated that HUVEC bind both purified [125I]HK-HC and [125I]HK-LC in a zinc(2+)-dependent manner and that HK-LC did not displace bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit the binding of HK to HUVEC. Our results, therefore, indicate that (1) HK is capable of binding to endothelial cells via both heavy and light chain moieties, (2) HKa has a higher affinity to HUVEC, and (3) purified heavy and light chains are capable of directly binding to HUVEC. The data are consistent with the presence of a single high-affinity site for HK on endothelial cells within which are subsites that bind to heavy and light chains.

scholarly journals Liver inositol, 1,4,5-trisphosphate-binding sites are the Ca2(+)-mobilizing receptors

1990 ◽  
Vol 270 (1) ◽  
pp. 227-232 ◽  
Author(s):  
D L Nunn ◽  
C W Taylor
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Maximal Effect ◽  
Equilibrium Conditions ◽  
Peripheral Tissues ◽  
High Affinity ◽  
Kd Values ◽  
Release Data ◽  
The Relationship ◽  
Similar Affinity

Ins(1,4,5)P3 is the intracellular messenger that in many cells mediates the effects of Ca2(+)-mobilizing receptors on intracellular Ca2+ stores. An Ins(1,4,5)P3 receptor from cerebellum has been purified and functionally reconstituted, but the relationship between this protein and the high-affinity Ins(1,4,5)P3-binding sites of peripheral tissues is unclear. We compared the Ins(1,4,5)P3-binding sites of liver and cerebellum by measuring inhibition of specific Ins(1,4,[32P]5)P3 binding by various ligands under equilibrium conditions, and find that each ligand binds with similar affinity in the two tissues. Earlier studies in which Ins(1,4,5)P3 binding and Ca2+ mobilization were measured under different conditions demonstrated large differences between KD values for binding and EC50 values (concn. giving half-maximal effect) for Ca2+ release. We show here that, when measured under identical conditions, KD and EC50 values for four agonists are similar. Schild analysis of inhibition of Ins(1,4,5)P3 binding by ATP demonstrates a competitive interaction between the two at the liver Ins(1,4,5)P3-binding site, and this partly accounts for earlier discrepancies in binding and Ca2(+)-release data. We conclude that the high-affinity Ins(1,4,5)P3-binding site of hepatocytes is likely to be the receptor that mediates Ca2+ mobilization, and that this receptor is at present indistinguishable from that in cerebellum.

scholarly journals Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1306-1311
Author(s):  
SR Reddigari ◽  
P Kuna ◽  
G Miragliotta ◽  
Y Shibayama ◽  
K Nishikawa ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Light Chain ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Light Chains ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
High Affinity ◽  
Umbilical Vein Endothelial Cells

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. Excision of the vasoactive peptide bradykinin by plasma kallikrein results in kinin-free HK that consists of a 65-Kd N-terminal heavy chain (HK-HC) linked to the C-terminal 45- Kd light chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor of SH-proteases and HK-LC contains the binding sites for coagulation cofactors prekallikrein and factor XI. HK has previously been shown to bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc(2+)-dependent manner by a single class of high-affinity binding sites. We have further characterized that interaction in order to determine the cell-binding regions of HK. Competition binding experiments have indicated that either HK-LC or HK-HC was able to inhibit the binding of labeled HK with a 50% inhibitory concentration (IC50) of 77 nmol/L and 89 nmol/L, respectively. Cleaved two-chain HK (HKa) had an IC50 of 73 nmol/L, whereas uncleaved HK had an IC50 of 335 nmol/L. Direct binding experiments have indicated that HUVEC bind both purified [125I]HK-HC and [125I]HK-LC in a zinc(2+)-dependent manner and that HK-LC did not displace bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit the binding of HK to HUVEC. Our results, therefore, indicate that (1) HK is capable of binding to endothelial cells via both heavy and light chain moieties, (2) HKa has a higher affinity to HUVEC, and (3) purified heavy and light chains are capable of directly binding to HUVEC. The data are consistent with the presence of a single high-affinity site for HK on endothelial cells within which are subsites that bind to heavy and light chains.

Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes

1988 ◽  
Vol 66 (11) ◽  
pp. 1238-1247
Author(s):  
Louise Brissette ◽  
Simon-Pierre Noël
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Rat Liver ◽  
Binding Sites ◽  
Apolipoprotein B ◽  
High Density Lipoproteins ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Liver Membranes

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.

scholarly journals A mono-sited transferrin from a representative deuterostome: the ascidian Pyura stolonifera (subphylum Urochordata)

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1047-1052 ◽  
Author(s):  
AW Martin ◽  
E Huebers ◽  
H Huebers ◽  
J Webb ◽  
CA Finch
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Large Fraction ◽  
Membrane Receptors ◽  
Iron Binding ◽  
Substantial Fraction ◽  
Color Formation ◽  
High Affinity ◽  
Iron Binding Protein ◽  
Rat Reticulocytes

An iron-binding protein has been found in the plasma of Pyura stolonifera. This protein has a molecular weight of about 41,000 +/- 2,000 and binds 1 mol iron/mol protein. The absorption maxima are lambda = 280 and lambda = 429 nm (E429/E280 = 0.044). Bicarbonate is bound concomitantly with high affinity and is necessary for optimal color formation at lambda = 429 nm. The protein showed a negligible exchange of iron with human apotransferrin under physiologic conditions over two hours. Upon incubation with rat reticulocytes, the protein reacts with membrane receptors for transferrins, and the protein, with its iron, is transported intracellularly where the iron is incorporated into heme. The 59Fe protein, after intravenous injection, disappears rapidly from the plasma and is excreted largely in the urine, with a substantial fraction present in the kidney and another large fraction present in the gut. These findings established the protein as a “transferrin” and support the concept that the larger transferrin molecule in vertebrates, with two iron-binding sites, resulted from a gene duplication.

scholarly journals Tuning the Properties of High Molecular Weight Chitosans to Develop Full Water Solubility Within a Wide pH Range

2020 ◽  
Author(s):  
Anderson Fiamingo ◽  
Sergio Paulo Campana Filho ◽  
Osvaldo Novais Oliveira Junior
Keyword(s):  
Molecular Weight ◽  
Biomedical Applications ◽  
Random Distribution ◽  
Water Solubility ◽  
Aqueous Media ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
H Nmr ◽  
Wide Ph Range ◽  
Ph Range

<p>The preparation of chitosans soluble in physiological conditions has been sought for years, but so far solubility in non-acidic aqueous media has only been achieved at the expense of lowering chitosan molecular weight. In this work, we applied the multistep ultrasound-assisted deacetylation process (USAD process) to β-chitin and obtained extensively deacetylated chitosans with high molecular weights (Mw ≥ 1,000,000 g mol<sup>-1</sup>). The homogeneous <i>N</i>-acetylation of a chitosan sample resulting from three consecutive USAD procedures allowed us to produce chitosans with a high weight average degree of polymerization (DPw ≈ 6,000) and tunable degrees of acetylation (DA from 5 to 80%). <i>N</i>-acetylation was carried out under mild conditions to minimize depolymerization, while preserving a predominantly random distribution of 2-amino-2-deoxy-D-glucopyanose (<i>GlcN</i>) and 2-acetamido-2-deoxy-D-glucopyanose (<i>GlcNAc</i>) units. This close to random distribution, inferred with deconvolution of nuclear magnetic resonance (<sup>1</sup>H NMR) spectra, is considered as responsible for the solubility within a wide pH range. Two of the highly <i>N</i>-acetylated chitosans (DA ≈ 60 % and ≈ 70 %) exhibited full water solubility even at neutral pH, which can expand the biomedical applications of chitosans. </p>

scholarly journals A mono-sited transferrin from a representative deuterostome: the ascidian Pyura stolonifera (subphylum Urochordata)

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1047-1052 ◽  
Author(s):  
AW Martin ◽  
E Huebers ◽  
H Huebers ◽  
J Webb ◽  
CA Finch
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Large Fraction ◽  
Membrane Receptors ◽  
Iron Binding ◽  
Substantial Fraction ◽  
Color Formation ◽  
High Affinity ◽  
Iron Binding Protein ◽  
Rat Reticulocytes

Abstract An iron-binding protein has been found in the plasma of Pyura stolonifera. This protein has a molecular weight of about 41,000 +/- 2,000 and binds 1 mol iron/mol protein. The absorption maxima are lambda = 280 and lambda = 429 nm (E429/E280 = 0.044). Bicarbonate is bound concomitantly with high affinity and is necessary for optimal color formation at lambda = 429 nm. The protein showed a negligible exchange of iron with human apotransferrin under physiologic conditions over two hours. Upon incubation with rat reticulocytes, the protein reacts with membrane receptors for transferrins, and the protein, with its iron, is transported intracellularly where the iron is incorporated into heme. The 59Fe protein, after intravenous injection, disappears rapidly from the plasma and is excreted largely in the urine, with a substantial fraction present in the kidney and another large fraction present in the gut. These findings established the protein as a “transferrin” and support the concept that the larger transferrin molecule in vertebrates, with two iron-binding sites, resulted from a gene duplication.

scholarly journals Evaluation of the Degree of Polymerization of the Proanthocyanidins in Cranberry by Molecular Sieving and Characterization of the Low Molecular Weight Fractions by UHPLC-Orbitrap Mass Spectrometry

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1504 ◽  
Author(s):  
Claudio Gardana ◽  
Paolo Simonetti
Keyword(s):  
Molecular Weight ◽  
Molecular Sieves ◽  
Degree Of Polymerization ◽  
Low Molecular Weight ◽  
Molecular Weight Distributions ◽  
Orbitrap Mass Spectrometry ◽  
Weight Distributions ◽  
Molecular Weight Fractions ◽  
Molecular Sieving

4-dimethylammino-cinnamaldehyde (DMAC) assays quantify total proanthocyanidins (PACs) but do not provide qualitative PAC molecular weight distribution information and cannot discriminate between A- and B-type PACs. We developed an efficient method for assessing PAC molecular weight distributions. The PACs from three commercial cranberry extracts (A1–A3) were fractionated by molecular sieves with cut-offs of 3, 10, 30, 50, and 100 kDa, and each fraction was analyzed by DMAC assays. A1, A2, and A3 contained 27%, 33%, and 15% PACs, respectively. Approximately 28 PACs, 20 flavonols, and 15 phenolic acids were identified by UHPLC-DAD-Orbitrap MS in A1 and A3, while A2 contained only flavan-3-ols. Epicatechin was the main monomer in A1 and A3, and catechin was the main in A2. Procyanidin A2 was the main dimer in A1 and A3, representing more than 85% of the total dimers, while it constituted approximately only 24% of A2. A1 and A3 contained quercetin, isorhamnetin, myricetin, and their glycosides, which were totally absent in A2. In A1 and A3 the PACs were mainly distributed in the fractions 30–3 and <3 kDa, while in A2 more than 70% were present in the fraction less than 3 kDa. Overall, obtained data strongly suggests that A2 is not cranberry-derived, or is adulterated with another source of PACs.

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