Alterations in high-affinity binding characteristics and levels of opioids in invertebrate ganglia during aging: Evidence for an opioid compensatory mechanism

1984 ◽  
Vol 4 (2) ◽  
pp. 143-155 ◽  
Author(s):  
Amy Chapman ◽  
Gonzalo Gonzales ◽  
Wayne R. Burrowes ◽  
Paul Assanah ◽  
Bernadette Iannone ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Compensatory Mechanism ◽  
High Affinity Binding ◽  
Binding Characteristics ◽  
High Affinity ◽  
Invertebrate Ganglia

scholarly journals Two Binding Sites for [3H]PBR28 in Human Brain: Implications for TSPO PET Imaging of Neuroinflammation

2010 ◽  
Vol 30 (9) ◽  
pp. 1608-1618 ◽  
Author(s):  
David R Owen ◽  
Owain W Howell ◽  
Sac-Pham Tang ◽  
Lisa A Wells ◽  
Idriss Bennacef ◽  
...  
Keyword(s):  
Binding Sites ◽  
Marked Reduction ◽  
Specific Binding ◽  
Translocator Protein ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Binding Characteristics ◽  
High Affinity ◽  
High Affinity Site ◽  
Site Binding

[11C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [11C]PK11195, another TSPO radioligand. We measured the specific binding signals with [3H]PK11195 and [3H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [3H]PBR28, although all samples showed [3H]PK11195 binding. There was a marked reduction in the affinity of [3H]PBR28 for TSPO in samples with no visible [3H]PBR28 autoradiographic signal ( K i=188±15.6 nmol/L), relative to those showing normal signal ( K i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [3H]PBR28 bound with a two-site fit in 40% of cases, with affinities ( K i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in Kd or Bmax for [3H]PK11195 in samples showing no [3H]PBR28 autoradiographic signal relative to those showing normal [3H]PBR28 autoradiographic signal. [3H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [11C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.

scholarly journals The overlapping octamer/TAATGARAT motif is a high-affinity binding site for the cellular transcription factors Oct-1 and Oct-2

1991 ◽  
Vol 277 (2) ◽  
pp. 541-545 ◽  
Author(s):  
C L Dent ◽  
D S Latchman
Keyword(s):  
Single Molecule ◽  
Affinity Binding ◽  
Heterologous Promoter ◽  
High Affinity Binding ◽  
Binding Characteristics ◽  
High Affinity ◽  
Octamer Motif ◽  
High Affinity Binding Site ◽  
Simplex Virus ◽  
Cellular Transcription

The octamer motif in cellular promoters and the related TAATGARAT element in the herpes simplex virus (HSV) immediate-early promoters can both bind cellular octamer-binding proteins. The overlapping octamer/TAATGARAT elements (consensus ATGCTAATGARAT) found in the HSV-1 IE1 promoter thus represent a composite motif, each portion of which can independently bind octamer-binding protein. By comparing the binding characteristics of this composite motif with its individual elements, we show that it binds a single molecule of either Oct-1 or Oct-2 with much higher affinity than does either an octamer or TAATGARAT motif alone. This strong binding allows this element to direct a much higher level of gene expression when linked to a heterologous promoter than that observed with each of its individual components.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

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.

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