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 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 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 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 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 Isolated rat hepatocytes acquire iron from lactoferrin by endocytosis

1995 ◽  
Vol 311 (2) ◽  
pp. 603-609 ◽  
Author(s):  
D D McAbee
Keyword(s):  
Binding Sites ◽  
Rat Hepatocytes ◽  
Iron Binding ◽  
C Cells ◽  
Dextran Sulphate ◽  
Isolated Rat Hepatocytes ◽  
Iron Binding Protein ◽  
Diferric Transferrin ◽  
By Products ◽  
Isolated Rat

The iron-binding protein lactoferrin (Lf) present in blood is metabolized by the liver. Isolated rat hepatocytes vigorously endocytose bovine Lf via recycling Ca2(+)-dependent binding sites, but the uptake of iron from Lf by hepatocytes has not been examined. In this study, isolated rat hepatocytes were incubated with radiolabelled bovine Lf (125I-Lf, 59Fe-Lf or 125I-59Fe-Lf) at 37 degrees C, then washed at 4 degrees C in the presence of dextran sulphate with either Ca2+ or EGTA to distinguish between total bound and internal radioactivity respectively. Cells internalized 125I-Lf protein and Lf-bound 59Fe at maximal endocytic rates of 1700 and 480 mol.cell-1.s-1 respectively. When Lf was normalized for 59Fe content, these endocytic rates were equivalent and reflected an uptake potential of at least 3400 mol of iron.cell-1.s-1. Cells prebound with 125I-59Fe-Lf to Ca2+(-)dependent sites at 4 degrees C internalized more than 80% of both 125I-Lf protein and Lf-bound 59Fe approx. 6 min after warming to 37 degrees C at similar rates (125I-Lf: k(in) = 0.276 min-1, 59Fe: k(in) = 0.303 min-1). Within 4 h at 37 degrees C, cells had released 25% or less internalized Lf protein in the form of acid-soluble 125I-by-products but retained all the Lf-delivered 59Fe. Hyperosmotic disruption of clathrin-dependent endocytosis blocked the uptake of 125I-Lf and Lf-bound 59Fe. Incubation of cells with 125I-59Fe-Lf and a 100 molar excess of diferric transferrin reduced slightly the endocytosis of 125I-Lf protein and 59Fe accumulation. Treatment of cells with the ferric chelator desferrioxamine did not alter uptake of 125I-Lf protein or Lf-bound 59Fe, but the ferrous chelator bathophenanthroline disulphonate slightly elevated endocytosis of 125I-Lf protein and Lf-bound 59Fe. These findings indicate that Lf does not release its bound iron before endocytosis. It was concluded from this study that hepatocytes take up iron from Lf at high rates by a process that requires endocytosis of Lf-iron complexes.

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 Cytosol intermediates in the transport of iron

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1051-1055 ◽  
Author(s):  
MT Nunez ◽  
ES Cole ◽  
J Glass
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Protein ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iron Binding ◽  
The Third ◽  
Initial Values ◽  
Iron Supply ◽  
Iron Binding Protein

Three 59Fe-labeled nonheme components of the cytosol were identified when rabbit reticuloyctes were incubated with 59Fe-labeled plasma under conditions in which the iron supply was not limiting. Two of these components were identified as ferritin and transferrin. The latter was characterized by gel filtration as having apparent molecular weight higher than transferrin, indicating that the transferrin may be complexed to another moiety. The third component, referred to as iron- binding protein-I (IBP-I), is as yet uncharacterized. When the reticulocytes were incubated with unlabeled plasma after pulse-labeling with 59Fe-labeled plasma, 59Fe radioactivity in these cytosol components decreased; after 15 min of chase, the 59Fe in ferritin, transferrin, and IBP-I fell to 64.6%, 26.5%, and 65.8% of the initial values, respectively. A good correlation existed between the decrease of 59Fe in these three nonheme compartments and the associated increase in 59Fe-heme. The data presented suggest that cytosol ferritin, transferrin, and IBP-I are intermediates in the transport of 59Fe from the plasma membrane to the mitochondria.

ʟ- Usnate-Urease Interactions: Binding Sites for the Ligand

1980 ◽  
Vol 35 (11-12) ◽  
pp. 1098-1100 ◽  
Author(s):  
Isabel García ◽  
Blanca Cifuentes ◽  
Carlos Vicente
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Binding Sites ◽  
Usnic Acid ◽  
Polymerization Process ◽  
High Affinity ◽  
High Molecular Weight Polymers

Abstract ʟ-usnic acid inactivates urease by formation of high molecular weight aggregates which can reached by a maximum of 880 000. ʟ-cysteine partially reverses the inactivation by stimulating the appearance of active high molecular weight polymers. The existence of two class of binding points for ʟ-usnic acid on the urease molecule is proposed, the first showing high affinity for the ligand, related with the loss of activity, and the second, of low affinity, related to polymerization process.

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