Two Distinctive Cell Binding Patterns by Vacuolating Toxin Fused with GlutathioneS-Transferase:  One High-Affinity m1-Specific Binding and the Other Lower-Affinity Binding for Variant m Forms†

Biochemistry ◽  
2001 ◽  
Vol 40 (39) ◽  
pp. 11887-11896 ◽  
Author(s):  
Wen-Ching Wang ◽  
Hung-Jung Wang ◽  
Chun-Hsien Kuo
Keyword(s):  
Specific Binding ◽  
The Other ◽  
Affinity Binding ◽  
Cell Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Vacuolating Toxin ◽  
Distinctive Cell

Somatotrophic receptors in hepatic tissue of the developing male pig

1989 ◽  
Vol 123 (1) ◽  
pp. 25-31 ◽  
Author(s):  
B. H. Breier ◽  
P. D. Gluckman ◽  
H. T. Blair ◽  
S. N. McCutcheon
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Plasma Concentrations ◽  
Age Groups ◽  
Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Igf I ◽  
High Affinity Binding Site ◽  
Scatchard Plots

ABSTRACT The development of hepatic somatotrophic receptors and plasma concentrations of insulin-like growth factor-I (IGF-I) were investigated at five different ages (2, 20, 35, 105 and 165 days) in four male pigs per group. The specific binding of 125I-labelled porcine GH (pGH) to hepatic somatotrophic membranes was very low at 2 days of age (0·53±0·12%), and increased progressively (P <0·01) with advancing age to 3·60 ± 0·95% at 165 days of age. Specific binding of 125I-labelled bovine GH (bGH) to the same membrane preparations was markedly higher than binding of 125I-labelled pGH; it also showed a distinct developmental increase (P <0·01) with age from 4·4 ± 0·55% at 2 days of age to 24·0±1·90% at 165 days of age. Plasma concentrations of IGF-I increased significantly (P <0·01) from 79 ± 14·0 μg/l at 2 days of age to 610 ± 64·0 μg/l at 165 days of age. Non-linear regression analysis of the competitive binding data using bGH as labelled and unlabelled ligands showed linear Scatchard plots in the three youngest age groups, with an association constant (Ka) of approximately 3·5 litres/nmol. Curvilinear Scatchard plots were observed in the two oldest age groups. The Ka for the higher affinity binding site (approximately 5·0 litres/nmol) was very similar to that for the sole site observed in the younger animals. The Ka of the lower affinity binding site was approximately 0·35 litres/nmol. There was a significant (P <0·01) developmental increase in the capacity of the higher affinity binding site from 12 ± 4·6 pmol/100 mg liver at 2 days of age to 91 ± 23·0 pmol/100 mg liver at 165 days of age. These studies demonstrate heterogeneity of somatotrophic hepatic membranes in the pig and show that low concentrations of a high affinity binding site are already present in newborn pigs. A considerable developmental increase was observed in the capacity of this binding site which correlated significantly (r = 0·82, P <0·01, n = 20) with plasma concentrations of IGF-I. The role of the lower affinity binding site which was observed in addition to the high affinity binding site in older pigs is less clear. The data from the present study support the hypothesis that the postnatal rise in plasma concentrations of IGF-I is associated with the developmental increase of the capacity of the high affinity somatotrophic receptors. Journal of Endocrinology (1989) 123, 25–31

Interactions of some partial agonists with high and low affinity binding sites in β-adrenoceptors

1987 ◽  
Vol 65 (1) ◽  
pp. 18-22 ◽  
Author(s):  
I. Takayanagi ◽  
K. Koike ◽  
A. Nakagoshi
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Specific Binding ◽  
The Other ◽  
Affinity Binding ◽  
High Affinity ◽  
Partial Agonists ◽  
Significant Difference ◽  
High Affinity Site ◽  
Derivatives Of

Interactions of derivatives of befunolol (BFE-37, BFE-55, and BFE-61), carteolol, and pindolol with β-adrenoceptors were tested in guinea pig isolated taenia caecum. All the drugs used acted as partial agonists on the β-adrenoceptors when compared with isoprenaline, a full agonist. The pA2 values of BFE-61, carteolol, and pindolol were significantly larger than their pD2 values, while there was no significant difference between the pA2 and pD2 values for BFE-37 and BFE-55. The specific binding of [3H]befunolol to microsomal fractions from the guinea pig taenia caecum distinguished two binding sites, high affinity and low affinity sites. Both sites are considered to be bound by 50 nM of [3H]befunolol. Specific 3H binding was displaced by BFE-61, carteolol, and pindolol in a biphasic manner but in a monophasic manner by BFE-37 and BFE-55. Furthermore, [3H]befunolol binding was only partially displaced by BFE-55 but completely displaced by the other drugs used. These results, together with our previous findings, suggest that BFE-61, carteolol, and pindolol discriminate between the two affinity binding sites in the β-adrenoceptors, which are not discriminated between by BFE-37, and further that BFE-55 may bind with only the high affinity site.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

scholarly journals DapE Can Function as an Aspartyl Peptidase in the Presence of Mn2+

2003 ◽  
Vol 185 (16) ◽  
pp. 4748-4754 ◽  
Author(s):  
Daniel H. Broder ◽  
Charles G. Miller
Keyword(s):  
Divalent Cations ◽  
The Other ◽  
Expression Vectors ◽  
Peptidase Activity ◽  
Native Protein ◽  
Lower Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Serovar Typhimurium ◽  
Dipeptidase Activity

ABSTRACT Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn2+. Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn2+-dependent peptidase is DapE (N-succinyl-l,l-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His6). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn2+-dependent aspartyl peptidase activity relative to the MPD dapE+ strain. In addition, purified DapE-His6 exhibited Mn2+-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn2+ in the high-affinity site and Mn2+ in the low-affinity site.

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

1987 ◽  
Author(s):  
J Mardiguian
Keyword(s):  
Aqueous Medium ◽  
Uronic Acid ◽  
Antithrombin Iii ◽  
Specific Binding ◽  
Factor Xa ◽  
Benzyl Ester ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Convincing Data

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

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

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.

scholarly journals Relations between high-affinity binding sites for l-tryptophan, diazepam, salicylate and Phenol Red on human serum albumin

1983 ◽  
Vol 209 (1) ◽  
pp. 135-142 ◽  
Author(s):  
U Kragh-Hansen
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Binding Sites ◽  
Association Constants ◽  
The Other ◽  
Affinity Binding ◽  
Phenol Red ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

Binding of L-tryptophan, diazepam, salicylate and Phenol Red to defatted human serum albumin was studied by ultrafiltration at pH 7.0. All ligands bind to one high-affinity binding site with association constants of the order of 10(4)-10(5)M-1. The number of secondary binding sites was found to vary from zero to five, with association constants about 10(3)M-1. Competitive binding studies with different pairs of the ligands were performed. Binding of both ligands was determined simultaneously. L-Tryptophan and diazepam were found to compete for a common high-affinity binding site on albumin. The following combinations of ligands do not bind competitively to albumin: L-tryptophan-Phenol Red, L-tryptophan-salicylate and Phenol Red-salicylate. On the other hand, high-affinity bindings of the three ligands do not take place independently but in such a way that binding of one of the ligands results in a decrease in binding of the other ligands. The decreases in binding are reciprocal and can be accounted for by introducing a coupling constant. The magnitude of the constant is dependent on the ligands being bound. In the present study, the mutual decrease in binding was more pronounced with L-tryptophan-salicylate and Phenol Red-salicylate than with L-tryptophan-Phenol Red.

Requirements for Specific Binding of Low Affinity Inhibitor Fragments to the SH2 Domain ofpp60Src Are Identical to Those for High Affinity Binding of Full Length Inhibitors

2003 ◽  
Vol 46 (24) ◽  
pp. 5184-5195 ◽  
Author(s):  
Gudrun Lange ◽  
Dominique Lesuisse ◽  
Pierre Deprez ◽  
Bernard Schoot ◽  
Petra Loenze ◽  
...  
Keyword(s):  
Specific Binding ◽  
Sh2 Domain ◽  
Full Length ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity
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