Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

1987 ◽  
Vol 65 (11) ◽  
pp. 2338-2345 ◽  
Author(s):  
Gordon T. Bolger ◽  
Kendall A. Marcus ◽  
Ronald Thibou ◽  
Phil Skolnick ◽  
Ben Avi Weissman
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Divalent Cations ◽  
Opiate Receptors ◽  
Opiate Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Regulatory Effects ◽  
In Vitro Effects

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5′-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity μ-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris–HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+,2.9 mM and Li, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the μ-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris–HCl. In 50 mM Tris–HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris–HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited, [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris–HCl, respectively. These findings indicate that the in vitro effects of Na+ at μ- and possibly other opiate receptors in rat brain are dependent on the concentration of Tris–HCl used in the assay buffer, lower concentrations of Tris-HCl revealing novel regulatory effects for Na+ at μ-opiate receptors.

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

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.

Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

1985 ◽  
Vol 63 (2) ◽  
pp. 128-136
Author(s):  
Deborah A. Brown ◽  
Robert A. Cook
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Binding Sites ◽  
Malic Enzyme ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Regulatory Effects ◽  
Metal Cofactors

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

High-affinity binding sites for 125I-labelled pancreatic secretory phospholipase A2 in rat brain

1997 ◽  
Vol 49 (1-2) ◽  
pp. 120-126 ◽  
Author(s):  
Kumlesh K Dev ◽  
Christian Foged ◽  
Heidi Andersen ◽  
Tage Honoré ◽  
Jeremy M Henley
Keyword(s):  
Phospholipase A2 ◽  
Rat Brain ◽  
Binding Sites ◽  
Affinity Binding ◽  
Secretory Phospholipase A2 ◽  
High Affinity Binding ◽  
Secretory Phospholipase ◽  
High Affinity

Rat brain membranes possess two high-affinity binding sites for [3H]somatostatin

1985 ◽  
Vol 55 (2) ◽  
pp. 161-166 ◽  
Author(s):  
D.R. Weightman ◽  
C.A. Whitford ◽  
C.R. Snell ◽  
B.H. Hirst ◽  
D.E. Brundish ◽  
...  
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Brain Membranes ◽  
Rat Brain Membranes

High affinity binding sites for a somatostatin-28 analog in rat brain

Life Sciences ◽  
1981 ◽  
Vol 28 (19) ◽  
pp. 2191-2198 ◽  
Author(s):  
Jean-Claude Reubi ◽  
Marilyn H. Perrin ◽  
Jean E. Rivier ◽  
Wylie Vale
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

1987 ◽  
Vol 65 (6) ◽  
pp. 1171-1181 ◽  
Author(s):  
Richard Larivière ◽  
Ernesto L. Schiffrin
Keyword(s):  
Rate Constant ◽  
Binding Sites ◽  
Divalent Cations ◽  
Dissociation Rate ◽  
Guanine Nucleotides ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Receptor Affinity ◽  
High Affinity ◽  
Binding Curves

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mg2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ > Co2+ > Ni2+ > Mg2+ > Ca2+ ≈ control without cations. Addition of Na+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 ± 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 ± 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, fmax and affinity were reduced. The addition of 100 μM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K−1). The equilibrium dissociation constant (KD) calculated with these rate constants (K−1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.

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