scholarly journals Integrin αv β8 Adopts a High Affinity State for Soluble Ligands Under Physiological Conditions

2017 ◽  
Vol 118 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Ping Hu ◽  
Bing-Hao Luo
Keyword(s):  
Physiological Conditions ◽  
High Affinity ◽  
Integrin Αv

scholarly journals Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

2012 ◽  
Vol 443 (1) ◽  
pp. 307-315 ◽  
Author(s):  
Linda Troeberg ◽  
Barbara Mulloy ◽  
Peter Ghosh ◽  
Meng-Huee Lee ◽  
Gillian Murphy ◽  
...  
Keyword(s):  
Sulfated Polysaccharide ◽  
Pentosan Polysulfate ◽  
Endogenous Inhibitor ◽  
Binding Ability ◽  
Physiological Conditions ◽  
Interaction Sites ◽  
High Affinity ◽  
Multiple Protein ◽  
Trimolecular Complex ◽  
Protein Interaction Sites

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

2007 ◽  
Vol 292 (6) ◽  
pp. C2204-C2212 ◽  
Author(s):  
Johan W. Jonker ◽  
Sandra Musters ◽  
Maria L. H. Vlaming ◽  
Torsten Plösch ◽  
Karin E. R. Gooijert ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Resistance Protein ◽  
Protoporphyrin Ix ◽  
Harderian Gland ◽  
Cancer Resistance ◽  
Erythropoietic Protoporphyria ◽  
Physiological Conditions ◽  
High Affinity ◽  
Expression Site ◽  
Resistance Protein

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria. This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias. In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1. Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins. Bcrp1−/− harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp1−/− liver. Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX. This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed.

Design and applications of methods for fluorescence detection of iron in biological systems

2002 ◽  
Vol 30 (4) ◽  
pp. 729-732 ◽  
Author(s):  
B. P. Espósito ◽  
W. Breuer ◽  
Z. I. Cabantchik
Keyword(s):  
Metal Binding ◽  
Biological Systems ◽  
Iron Chelator ◽  
Specific Cell ◽  
Iron Binding ◽  
Physiological Conditions ◽  
High Affinity ◽  
Cell Compartments ◽  
Heterogeneous Nature ◽  
Labile Iron

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.

Glutamate receptor binding to cat central nervous system membranes

1980 ◽  
Vol 58 (7) ◽  
pp. 534-538 ◽  
Author(s):  
Richard A. Head ◽  
Godfrey Tunnicliff ◽  
G. Keith Matheson
Keyword(s):  
Spinal Cord ◽  
Binding Sites ◽  
Cervical Spinal Cord ◽  
Specific Binding ◽  
Amygdaloid Complex ◽  
Sodium Ions ◽  
Physiological Conditions ◽  
High Affinity ◽  
Glutamate Binding ◽  
Structural Analogues

L-[3H]Glutamate exhibited specific binding to fresh membranes of cat CNS under physiological conditions of pH and temperature. This binding occurred in the absence of sodium ions. Kinetic analysis of the data for cerebellum suggested the presence of two distinct binding sites: a high-affinity process (Kd = 0.33 μM) with a capacity of 15 pmol/mg protein and a low-affinity process (Kd = 1.8 μM) which had a capacity of 65 pmol/mg protein. Several structural analogues of glutamic acid were able to appreciably inhibit the binding of [3H]glutamate. The distribution of glutamate binding between 12 regions of the CNS was measured. The amygdaloid complex exhibited the highest binding followed by hippocampus > hypothalamus ≡ visual cortex ≡ thalamus ≡ caudate nucleus > olfactory bulb ≡ tectum ≡ cerebellum > dorsal pons ≡ medulla > cervical spinal cord. These findings are consistent with the binding of [3H]glutamate being to its receptor.

scholarly journals Metabolism of acetylcholine in the nervous system of Aplysia californica. II. Reginal localization and characterization of choline uptake.

1975 ◽  
Vol 65 (3) ◽  
pp. 275-291 ◽  
Author(s):  
M L Eisenstadt ◽  
S N Treistman ◽  
J H Schwartz
Keyword(s):  
Nervous System ◽  
Nervous Tissue ◽  
Aplysia Californica ◽  
Nerve Terminals ◽  
Choline Uptake ◽  
Physiological Conditions ◽  
High Affinity ◽  
Complicated Manner ◽  
Cholinergic Cell

The choline required for synthesis of acetylcholine is derived exogenously by Aplysia ganglia. Under physiological conditions choline was taken up primarlily by neuropile and nerves and not by cholinergic cell bodies. In addition, compared with their contents of choline acetyltransferase, those components of nervous tissue which contain nerve terminals and axons synthesized acetylcholine far more efficiently. Choline was accumulated by high and low affinity uptake processes; the high affinity process appeared to be characteristic of cholinergic nuerons (Swartz, J. H., M. L. Eisenstadt, and H. Cedar.1975. J. Gen. Physiol. 65:255). The two uptake processes were similarly affected by temperature with a Q10 of 2.8. Both were dependent on a variety of ions in a complicated manner. High affinity uptake seemed to be more dependent on Na+, showed greater inhibition by ouabain, and was selectively inhibited by oxotremorine. We found that the functional state of neurons did not alter uptake of radioactive choline by either process, nor did it change the conversion to radioactive acetylcholine.

scholarly journals Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

1970 ◽  
Vol 120 (4) ◽  
pp. 831-836 ◽  
Author(s):  
J. Méšter ◽  
D. M. Robertson ◽  
Patricia Feherty ◽  
A. E. Kellie
Keyword(s):  
Dissociation Constant ◽  
Oestrogen Receptor ◽  
Scatchard Plot ◽  
Assay Method ◽  
Physiological Conditions ◽  
High Affinity ◽  
Receptor Sites ◽  
Assay Procedure ◽  
Order Of Magnitude

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k−1) and low-affinity (k−2) receptor sites and [3H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.

Multivalent DNA Aptamer-Based Therapeutic Agents for Lymphoma and Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2711-2711
Author(s):  
Prabodhika Mallikaratchy ◽  
Alessandro Ruggiero ◽  
William Maguire ◽  
Kelly Piersanti ◽  
Jeffrey Gardner ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
B Cell ◽  
Cell Lines ◽  
Drug Carrier ◽  
Original Version ◽  
Physiological Conditions ◽  
High Affinity ◽  
Nuclease Resistance ◽  
Membrane Bound

Abstract Abstract 2711 Poster Board II-687 Non-Hodgkins lymphomas affect 450,000 patients in the United States and even with recent advances in antibody-based therapies; more than 20,000 people will die of their disease annually. The goal of this work was to develop a high affinity, stable aptamer selective for B-cell leukemias and lymphomas. Aptamers are small DNA molecules that have the ability to bind to proteins with high affinity and specificity. They are also ideal candidates as therapeutic carriers. Aptamer binding is based on the ability of small oligonucleotide polymers (typically 20–80mers) to fold into unique three-dimensional structures that can interact with a specific target. Based on nature of this interaction, aptamers could be considered to be antibody analogs. Compared to antibodies however, one of the inherent properties of aptamers is that their small size (typically 10–20,000 daltons for aptamers vs 150,000 daltons for antibodies,) might address some difficult pharmacologic issues of antibodies, which penetrate slowly into tumors and clear the blood slowly. Recently, the TDO5 aptamer was identified and it was found to bind to membrane bound human IgM, a component of the BCR complex in B-Cell neoplasms. In contrast to currently available monoclonal antibodies, TD05 binds to membrane bound human IgM only and not with soluble IgM, eliminating the possibility of competitive inhibition by soluble IgM in the serum. The specificity of the aptamer was confirmed by screening with 24 cell lines and fresh clinical leukemia samples. Out of 24 cell lines, the IgM-negative cells, including T-cell leukemias and solid tumor lines such as breast, kidney, and colon and ovarian, showed no binding with TD05 indicating there is no non-specific adhesion with cell lines. One of the challenges of using the current form of TD05 as a drug carrier is that it is not yet suitable for use in vivo because of low avidity (>10uM) and stability (t ½= 1min) at physiological conditions. In order to increase the affinity of this aptamer, a new truncated multivalent and nuclease stable aptamer was designed. First, truncation of the original version of TD05 was considered because reduced size may lead to more efficient chemical syntheses and better pharmacologic properties. The resulting TD05.1 has a 5-fold increased affinity compared to original version of TD05. Second, bivalent (BV) aptamers utilizing TD05.1 with various polyethylene glycol (PEG) linker lengths were designed. Linker length is critical in designing multivalent aptamers to avoid loss of binding due to steric hindrance and to optimize the binding geometry, both of which would affect binding affinity. An optimal linker length of ∼16nm was chosen after empiric binding studies. Nuclease stability was also addressed by the introduction of chemical functionalities into TD05.1. The introduction of non-natural bases such as LNA bases (Locked Nucleic Acids), have been demonstrated to be effective in this regard. The stability of LNA stems from the bicyclic furanose unit locked in a sugar conformation. In order to retain the specific recognition and 3-dimensional nature of the aptamer's folding, LNA analogues were only introduced at the regions that are not involved in binding. Additionally, to further improve nuclease resistance, increase circulation time in vivo and to prevent non-specific adhesion to serum proteins and cells, the LNA modified BV aptamer TD05.1 was modified with polyethylene glycol at the 3' and 5' ends. The introduction of LNA and polyethylene glycol further stabilized the secondary structure, increasing the affinity and nuclease resistance in serum from one minute to seven hours(t ½> 420 min). We then constructed a trivalent analog of the aptamer with multiple functionalities, including chelators and fluorophores, which showed ∼25-fold higher affinity compared to monomeric aptamer at physiological conditions. In conclusion, this study demonstrates the development of a trivalent, high affinity aptamer selective for the membrane bound human IgM found in B-cell leukemias and lymphoma. Studies to assess the biological activity and the use of this construct as a drug carrier to treat B-NHL and B-CLL are in progress. Disclosures: No relevant conflicts of interest to declare.

scholarly journals What ATP binding does to the Ca2+pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2+

2020 ◽  
Vol 117 (31) ◽  
pp. 18448-18458 ◽  
Author(s):  
Yoshiki Kabashima ◽  
Haruo Ogawa ◽  
Rie Nakajima ◽  
Chikashi Toyoshima
Keyword(s):  
Crystal Structure ◽  
Cardiac Muscle ◽  
Binding Sites ◽  
Phosphorylation Site ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Physiological Conditions ◽  
High Affinity ◽  
Ph Values ◽  
P Domain

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

Nanomolar affinity protein trans-splicing monitored in real-time by fluorophore–quencher pairs

2017 ◽  
Vol 53 (3) ◽  
pp. 545-548 ◽  
Author(s):  
M. Braner ◽  
R. Wieneke ◽  
R. Tampé
Keyword(s):  
Real Time ◽  
Protein Labeling ◽  
Online Detection ◽  
Physiological Conditions ◽  
High Affinity ◽  
Cellular Environment ◽  
Trans Splicing

We combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal ‘traceless’ protein labeling at nanomolar concentrations under physiological conditions in cellular environment.

scholarly journals Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, pregnancy, maturation and after ovariectomy

1970 ◽  
Vol 120 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Patricia Feherty ◽  
D. M. Robertson ◽  
H. B. Waynforth ◽  
A. E. Kellie
Keyword(s):  
Early Pregnancy ◽  
Similar Pattern ◽  
Quantitative Method ◽  
Oestrous Cycle ◽  
High Concentration ◽  
Physiological Conditions ◽  
High Affinity ◽  
Receptor Sites ◽  
Receptor Concentration ◽  
Cyclic Changes

A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.

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