Imaging of the super high affinity binding sites for [3H]Ro15–4513 in rat hippocampus: comparison between in vitro and in vivo binding

1998 ◽  
Vol 250 (3) ◽  
pp. 161-164 ◽  
Author(s):  
Takayuki Nakano ◽  
Toshiyuki Satoh ◽  
Keiji Mori ◽  
Osamu Inoue
Keyword(s):  
Binding Sites ◽  
Rat Hippocampus ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
In Vivo Binding ◽  
High Affinity

scholarly journals Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

2000 ◽  
Vol 182 (14) ◽  
pp. 4022-4027 ◽  
Author(s):  
Richard A. Fekete ◽  
Laura S. Frost
Keyword(s):  
Binding Sites ◽  
Integration Host Factor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Negative Dominance ◽  
Transfer Apparatus

ABSTRACT Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage atnic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage atnic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriTallowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

Zinc Trafficking 1. Probing the Roles of Proteome, Metallothionein, and Glutathione

Metallomics ◽  
2021 ◽  
Author(s):  
Afsana Mahim ◽  
Mohammad Mahim ◽  
David H Petering
Keyword(s):  
Carbonic Anhydrase ◽  
Binding Sites ◽  
Affinity Binding ◽  
Conditional Stability ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Conditional Stability Constant ◽  
High Affinity Binding ◽  
High Affinity

Abstract The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that non-specific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione. In agreement, inclusion of glutathione accelerated the reaction in a concentration-dependent manner. The implications of abundant high affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with glutathione in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

2006 ◽  
Vol 188 (6) ◽  
pp. 2081-2095 ◽  
Author(s):  
Leah S. Feldman-Cohen ◽  
Yongping Shao ◽  
Derrick Meinhold ◽  
Charmi Miller ◽  
Wilfredo Colón ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Motif ◽  
Affinity Binding ◽  
Flanking Sequence ◽  
Binding Assays ◽  
High Affinity Binding ◽  
High Affinity

ABSTRACT Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd , ∼1 to 4 nM) and nonspecific (Kd , ∼4 μM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and λ attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the λ attR Fis site, and the role of R89 was dramatically altered by the λ attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

EFFECT OF FUROSEMIDE ON PLATELET AGGREGATION AND ON ALPHA-ADRENOCEPTOR-DENSITY IN MAN.

1987 ◽  
Author(s):  
A Kribben ◽  
E Fritschka ◽  
M Sibold ◽  
M Fassbender ◽  
A Distler ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Affinity Binding ◽  
Pressor Effect ◽  
High Affinity Binding ◽  
Alpha Adrenoceptor ◽  
High Affinity

Furosemide (FUR) reduces the ∝2-adrenoceptor- (∝2-R) mediated pressor effect of norepinephrine. Since it has been shown that FUR reduces platelet aggregation ( AGG) we studied the effect of FUR on α2-R as well as that on the ∝2-R-medi ated epinephrine ( EPI)-induced AGG a) ex vivo and b) in vitro. For comparison the effect of FUR on ADP-induced AGG was also studied. Methods: a) 8 normotensive men received FUR (30 mg b. i.d. ) for 3 weeks. EPI- (1 μmol/1) and ADP- (1 μmol/1) induced platelet AGG were measured before as well as after 3 weeks of FUR application with a semiautomatic device ( APACT) . Platelet ∝2-R-density was measured by 3H-yohimbine-binding and the fraction of high-affinity binding-sites was measured by competi tion of 3H-yohimbine with EPI. b) Platelet-rich plasma was incubated with FUR (1 mmol/1) for 10 min at 37 °C and AGG was measured (n=7) as described. Results: a) FUR decreased ∝2-R-densi ty from 293± 28 to 251±; 21 fmol/mg protein (p< 0.01) and high affinity binding sites from 64± 3 to 55± 5 % (p< 0.01). FUR inhibited ADP-induced AGG ex vivo while EPI-induced AGG did not change (table). b) In vitro FUR inhibited total ADP-induced AGG from 7 4 ± 4 to 45± 8 % (p< 0. 01) while again EPI-induced AGG did not change.Conclusions: The results suggest separate effects of FUR on α2 R-density and on platelet AGG. Although FUR decreased < r2-R, EPI-induced platelet AGG remained unchanged. Since FUR inhibited ADP-induced AGG ex vivo as well as in vitro FUR may interfere directly with the mechanism of ADP-induced platelet AGG.

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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