Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead

1994 ◽  
Vol 140 (2) ◽  
pp. 217-227 ◽  
Author(s):  
I Navarro ◽  
T W Moon
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Insulin Binding ◽  
American Eel ◽  
Brown Bullhead ◽  
Teleost Species ◽  
High Affinity ◽  
Glucagon Like Peptide 1 ◽  
High Concentrations

Abstract We have characterized the specific binding of glucagon in hepatocytes isolated from two teleost species, the American eel (Anguilla rostrata) and the brown bullhead (Ictalurus nebulosus). Specific glucagon binding was 9·3 and 10·7% in bullhead and eel hepatocytes respectively, after a 2-h incubation at 12 °C. Curvilinear Scatchard plots suggest the presence of two classes of binding sites with apparent dissociation constants (Kd) of 1·97 nm (high affinity) and 17·3 nm (low affinity) for bullhead and 2·68 and 22·9 nm for eel cells. The number of high-affinity binding sites per cell was significantly higher in the eel (10 413) than in the bullhead (3811). The number of high-affinity insulin-binding sites was approximately two times higher than that for glucagon in bullheads and the opposite in the eel hepatocytes. In competition experiments, insulin did not displace 125I-labelled glucagon binding in the hepatocytes of either species, while glucagon-like peptide-1(7–37) (GLP-1) displaced glucagon but only at high concentrations, suggesting separate glucagon- and GLP-1-binding sites. The rate of dissociation of hepatocyte-bound 125I-labelled glucagon was similar for both species. Preincubation of hepatocytes in 100 nm glucagon decreased the number of high-affinity glucagon-binding sites by approximately 55% in both species, while the Kd values remained unchanged. Glucagon bound to the cell surface is internalized by fish hepatocytes. These properties indicate that the glucagon binding to hepatocytes of these two teleost species is similar to that reported for mammalian hepatocytes. Journal of Endocrinology (1994) 140, 217–227

scholarly journals Characterization of calcium binding to brush-border membranes from rat duodenum

1982 ◽  
Vol 208 (3) ◽  
pp. 773-781 ◽  
Author(s):  
A Miller ◽  
S T Li ◽  
F Bronner
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Relative Effectiveness ◽  
Brush Border Membranes ◽  
High Affinity ◽  
High Affinity Site ◽  
Rat Duodenum ◽  
High Concentrations

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.

THE BINDING OF INSULIN AND SOMATOMEDIN A TO HUMAN PLACENTAL MEMBRANE

1975 ◽  
Vol 80 (1) ◽  
pp. 14-31 ◽  
Author(s):  
K. Takano ◽  
K. Hall ◽  
L. Fryklund ◽  
A. Holmgren ◽  
H. Sievertsson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Specific Binding ◽  
Insulin Binding ◽  
Radioreceptor Assay ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Mean Values ◽  
High Affinity

ABSTRACT A particulate membrane fraction from human placental membrane was shown to be rich in binding sites not only for insulin but also for somatomedin A. The binding of the 125I-labelled peptide was time and temperature dependent. Degrading activity present in the membrane fraction was negligible at +4°C. The Scatchard plot for insulin binding revealed two types of binding sites with an apparent high affinity constant of 3.8× 108 m−1 and with 5.4 × 10−9 moles of binding sites per mg of membrane protein. The Scatchard analysis of somatomedin A revealed two classes of binding sites with an apparent high affinity constant of 2.7 × 107 m−1 and with 1.9× 10−8 moles of binding sites per mg of membrane protein. In high concentrations insulin interfered with the specific binding sites for somatomedin A and vice versa. In comparison with insulin the somatomedin A preparation was one million times more potent in displacing labelled somatomedin A than in displacing labelled insulin from their respective binding sites. A radioreceptor assay utilizing particulate placental membrane and labelled somatomedin A purified on the membrane enabled the determination of somatomedin in unextracted serum. The mean values of somatomedin A in sera from patients with pituitary dwarfism and acromegaly were 0.57 and 3.2 U/ml, respectively by radioreceptor assay and 0.41 and 1.61 U/ml, respectively by bioassay. Various causes of this discrepancy between the methods are discussed.

The identification of 125I-labelled iodomelatonin-binding sites in the testes and ovaries of the chicken (Gallus domesticus)

1992 ◽  
Vol 133 (1) ◽  
pp. 5-11 ◽  
Author(s):  
E. A. Ayre ◽  
H. Yuan ◽  
S. F. Pang
Keyword(s):  
Acetic Acid ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Testicular Tissue ◽  
Equilibrium Studies ◽  
Middle Point ◽  
High Affinity ◽  
Middle Range

ABSTRACT The existence of 125I-labelled iodomelatonin-binding sites in chicken ovaries and testes was investigated. The specific binding of 125I-labelled iodomelatonin to chicken ovarian and testicular tissue satisfies all the criteria for a binding site. It was rapid, stable, saturable, reversible, specific and of high affinity. Equilibrium studies showed that total and non-specific binding increased over a range of 5–150 pmol 125I-labelled iodomelatonin/1 tested, with specific binding reaching saturation towards the middle range of radioligand concentrations. Scatchard analyses indicated a dissociation constant (Kd) of 36·5 ± 5·3 pmol/l (means ± s.e.m.) in the membrane preparations of chicken testes at the middle point in the period of light and a maximum number of binding sites (Bmax) of 0·93 ±0·40 fmol/mg protein (n = 6). In membrane preparations of chicken ovaries, the Kd was 102·2 ± 27·3 pmol/l and the Bmax was 2·77± 0·38 fmol/mg protein (n= 6). Equilibrium and kinetic dissociation constants in the picomolar range indicate high-affinity and physiologically relevant 125I-labelled iodomelatonin-binding sites. Competitive inhibition studies determined the following order of relative potency for inhibition of 125I-labelled iodomelatonin-binding to chicken gonadal membranes: 6-chloromelatonin > melatonin > N-acetylserotonin > > > 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, l-acetylindole-3-acetic acid, 5-hydroxyindole-3-acetic acid and l-tryptophan. The presence of 125I-labelled iodomelatonin-binding sites suggests a direct pinealgonadal connection in the chicken. Journal of Endocrinology (1992) 133, 5–11

Chronic treatment with bovine growth hormone upregulates high-affinity hepatic somatotropic receptors in sheep

1991 ◽  
Vol 124 (3) ◽  
pp. 307-313 ◽  
Author(s):  
Helga Sauerwein ◽  
Bernhard H. Breier ◽  
John J. Bass ◽  
Peter D. Gluckman
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Hormone Treatment ◽  
Bovine Growth Hormone ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Gh Therapy ◽  
High Affinity

Abstract. We evaluated the effect of chronic bovine growth hormone treatment on the hepatic somatotropic receptor. Growing lambs were treated with bGH at 0, 0.05, 0.15, 0.25 or 0.55 mg · kg−1 · day−1 daily (N=5/group) for 56 days. The binding of ovine GH to hepatic membranes washed with 4 mol/l MgCl2 and prepared in the presence of aprotinin was examined. The specific binding of oGH was increased (p<0.01) from 7.1±1.2% in saline-treated controls to 17.4±1.5% in the 0.55 mg · kg−1 · day−1 group. Scatchard analysis showed curvilinear plots that best fitted a two-site model in 22/25 livers. The two sites had estimated dissociation constants (Kd) of 3 to 13 nmol/l for the low-affinity site and a Kd ranging from 0.17 to 0.31 nmol/l for the high-affinity site. Treatment with bGH had no consistent effect on the affinity of either binding site. However, bGH therapy was associated with a dose-dependent increase (p<0.01) in the number of high-affinity somatotropic receptors. There was no effect of bGH therapy on the concentration of low-affinity binding sites. The concentration of high-affinity receptors correlated with weight gain (r=0.54, p<0.01), fat content (r=−0.54, p<0.01), protein content (r=0.40, p<0.05), and plasma IGF-I (r=0.57, p<0.005). The concentrations of low-affinity binding sites showed no such correlations. These observations demonstrate that an important effect of chronic GH therapy in animals with an intact somatotropic axis is to increase significantly the concentration of high-affinity somatotropic receptors. It is suggested that enhancement of the number of high-affinity somatotropic receptors is central to the determination of the efficacy of GH therapy.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

scholarly journals Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

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