Mucosal gastrin receptor. II. Physical characteristics of binding.

1979 ◽  
Vol 237 (3) ◽  
pp. E295
Author(s):  
K Takeuchi ◽  
G R Speir ◽  
L R Johnson
Keyword(s):  
Binding Sites ◽  
Low Temperatures ◽  
Specific Activity ◽  
Specific Binding ◽  
Cooperative Effect ◽  
Trypsin Treatment ◽  
Single Class ◽  
Gastrin Receptor ◽  
Protein Nature

Gastrin binding to rat gastric mucosal membrane preparations revealed a linear Scatchard plot, suggesting the existence of a single class of binding sites. Further studies revealed the absence of a positive cooperative effect. Because the system appeared to satisfy the conditions for Michaelis-Menten type kinetics, the determination of a Kd of approximately 3.6 X 10(-10) M was justified. These studies also demonstrated the efficacy of the specific activity of our iodinated gastrin. Trypsin treatment abolished specific binding of gastrin to the membrane preparation, indicating the protein nature of the receptor. The importance of maintaining extremely low temperatures during the incubation and wash period is demonstrated by the decrease in both the rate and the amount of dissociation at 0 degrees C compared with 30 degrees C.

scholarly journals An Isotopic Method for the Determination of Vitamin B12 Levels in Blood

Blood ◽  
1963 ◽  
Vol 21 (1) ◽  
pp. 70-79 ◽  
Author(s):  
RUSSEL M. BARAKAT ◽  
ROGER P. EKINS
Keyword(s):  
Vitamin B12 ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Binding ◽  
Normal Subjects ◽  
Free Form ◽  
Protein Binding Sites ◽  
Isotopic Method ◽  
Specific Binding Protein

Abstract Vitamin B12 is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation of protein binding sites and excess vitamin remains in free form. The ratio of free to bound fractions thus quantitatively depends upon the concentration of exogenous compound. This observation has been utilized to determine amounts of vitamin B12 extracted from the blood of normal subjects and of patients with certain pathologic conditions. The method is simple and reproducible. The sensitivity of the method is such that vitamin levels down to roughly 20 µµg./ml. may be evaluated using labeled vitamin B12 of a specific activity of about 1 µc./µg. Repeated assays on identical specimens of normal plasma have shown a reproducibility of about 5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml. with an average of 611 ± 167. Values observed in plasma taken from patients suffering from pernicious anemia were around 100 µµg./ml. or less. Results on subjects with other pathologic conditions are also presented and the limitations of the method are discussed.

Dissociation of contraction and muscarinic receptor binding to isolated smooth muscle cells

1986 ◽  
Vol 251 (4) ◽  
pp. G546-G552 ◽  
Author(s):  
S. M. Collins ◽  
D. J. Crankshaw
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Muscle Cells ◽  
Muscarinic Agonists ◽  
Isolated Cells ◽  
Single Class ◽  
Antagonist Binding ◽  
Isolated Smooth Muscle Cells

We examined changes in [3H]QNB binding and cell length induced by muscarinic ligands in a suspension of single smooth muscle cells isolated from the canine stomach. Cells contracted following a brief (30 s) exposure to picomolar concentrations of muscarinic agonists and yielded ED50 values of 1.0 +/- 0.7 pM for oxotremorine, 12.5 +/- 1.8 pM for carbachol, and 16.0 +/- 2.9 pM for metacholine. Contraction was inhibited by atropine with a pA2 value of 10.2 +/- 1.1. The binding of [3H]QNB was rapid and reversible and was stereospecific and pharmacologically appropriate. Specific binding of [3H]QNB was saturable and bound with high affinity (KD 1.04 +/- 0.23 nM) to a single class of sites, of which there were approximately 200,000/cell. In competition experiments antagonist binding was generally homogeneous, whereas that of agonists was heterogeneous and subpopulations of binding sites with different affinities for agonists were identified. The Ki value of 8.1 +/- 1.1 nM for inhibition of QNB binding by atropine was greater than the pA2 of 10.2 +/- 1.1 derived from contraction studies. Furthermore, whereas picomolar concentrations of agonists induced cell contraction, substantially higher concentrations (10 nM to 10 mM) were required to inhibit [3H]QNB binding to the isolated cells.

Evidence for a direct action of GH on haemopoietic cells of a marine fish, the gilthead sea bream (Sparus aurata)

1995 ◽  
Vol 146 (3) ◽  
pp. 459-467 ◽  
Author(s):  
J A Calduch-Giner ◽  
A Sitjà-Bobadilla ◽  
P Álvarez-Pellitero ◽  
J Pérez-Sánchez
Keyword(s):  
Binding Sites ◽  
Sparus Aurata ◽  
Binding Capacity ◽  
Specific Binding ◽  
Head Kidney ◽  
Gilthead Sea Bream ◽  
Sea Bream ◽  
Single Class ◽  
Lower Vertebrate

Abstract Receptors for GH were characterized in the head kidney of gilthead sea bream (Sparus aurata), using radioiodinated and biotinylated ligands. The specific binding of radiolabelled recombinant gilthead sea bream GH (rsbGH) to head kidney membrane preparations was dependent on membrane concentration. Salmon prolactin, salmon gonadotrophin and carp gonadotrophin did not compete for 125I-labelled rsbGH-binding sites. Unlabelled rsbGH competitively displaced 125I-labelled rsbGH bound to head kidney membranes. Scatchard plots were always linear, denoting the presence of a single class of binding sites. The binding affinity (Ka=2·7 × 109 m−1) was equivalent to that found in liver membrane preparations, but the binding capacity (2·5 ±0·30 fmol/mg protein) was 50- to 75-fold lower. To identify the cells which express the GH receptor, head kidney smears were incubated with biotinylated rsbGH, followed by incubation with an avidin–biotin complex conjugated to alkaline phosphatase. The reaction with the new-fuchsin substrate gave a red precipitate, showing a specific and intense labelling in erythroblasts, polychromatophilic erythroblasts and myeloblasts. Noticeable binding was observed in myelocytes and immature granulocytes, tending to disappear at the latter stages of granulocyte maturation. Light but appreciable binding was also observed in monocytes, lymphocytes and acidophilic erythroblasts, whereas it was completely absent in proerythrocytes and erythrocytes. The proliferative action of rsbGH and recombinant human IGF-I on in vitro cultures of head kidney cells was demonstrated by a 5-bromo-2′-deoxy-uridine immunoassay. To our knowledge, this is the first report that provides suitable evidence for a role of GH as a haemopoietic growth and differentiation factor in lower vertebrate species. Journal of Endocrinology (1995) 146, 459–467

Determination of specific activity of labeled ligands by nonlinear regression analysis

1993 ◽  
Vol 265 (5) ◽  
pp. E728-E735 ◽  
Author(s):  
J. A. Durr
Keyword(s):  
Regression Analysis ◽  
Nonlinear Regression ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Nonlinear Regression Analysis ◽  
Independent Variables ◽  
Lysine Vasopressin ◽  
Dependent Variables

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells

1992 ◽  
Vol 9 (2) ◽  
pp. 103-114 ◽  
Author(s):  
C. L. Broadhead ◽  
U. T. O'Sullivan ◽  
C. F. Deacon ◽  
I. W. Henderson
Keyword(s):  
Sodium Chloride ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Binding Sites ◽  
Specific Binding ◽  
Anguilla Anguilla ◽  
Chloride Cells ◽  
Dependent Manner ◽  
Single Class ◽  
Atrial Natriuretic

ABSTRACT The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in freshwater (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.

Specific binding of vasoactive intestinal peptide to human circulating mononuclear cells

1983 ◽  
Vol 61 (7) ◽  
pp. 664-671 ◽  
Author(s):  
C. A. Ottaway ◽  
C. Bernaerts ◽  
B. Chan ◽  
G. R. Greenberg
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
Mononuclear Cells ◽  
Specific Binding ◽  
Maximal Effect ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Peptide Interactions ◽  
Tracer Dilution ◽  
Dose Dependent

The interaction of the neuropeptide vasoactive intestinal peptide with human circulating mononuclear cells has been studied. Mononuclear cells were able to bind radiolabelled vasoactive intestinal peptide; the binding was rapid, reversible, saturable, and specific for vasoactive intestinal peptide. A fragment of vasoactive intestinal peptide (10–28) was 25-fold less effective than intact peptide as a competitor for the binding of the tracer. Secretin was 100-fold less effective as a competitor and glucagon competed poorly even at concentrations 10 000 times greater than that of the tracer molecule. In tracer dilution studies, the binding suggested a single class of binding sites with an apparent dissociation constant (Kd) of 2.4 × 10−10 M and a capacity of 2 000 sites per cell. In the presence of vasoactive intestinal peptide, there was a dose-dependent augmentation of cyclic AMP in the mononuclear cells. The concentration of vasoactive intestinal peptide which produced a half-maximal effect was the same as the Kd for the peptide binding. We conclude that mononuclear cells have specific high-affinity binding sites for vasoactive intestinal peptide. Interactions between mononuclear cells and vasoactive intestinal peptide may be an important mechanism modulating local immune responses within tissues innervated by vasoactive intestinal peptide containing neurons.

The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling

2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Specific Binding ◽  
Adrenergic System ◽  
Camp Production ◽  
Binding Assays ◽  
Single Class ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Adrenergic Antagonist ◽  
Number Of Binding Sites

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.

Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.

1980 ◽  
Vol 26 (6) ◽  
pp. 718-723
Author(s):  
F H Verhoff ◽  
P J Lisi ◽  
C D Fischer ◽  
J W Teipel ◽  
G Goldstein ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Binding Constants ◽  
Experimental Information ◽  
Antibody Binding ◽  
Displacement Analysis ◽  
Additional Information ◽  
Graphical Technique ◽  
Materials Used

Abstract A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.

scholarly journals Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 761-776 ◽  
Author(s):  
FM Uckun ◽  
DE Myers ◽  
AS Fauci ◽  
M Chandan-Langlie ◽  
JL Ambrus
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphoblastic Leukemia ◽  
Interleukin 1 ◽  
Receptor Occupancy ◽  
Binding Assays ◽  
Single Class ◽  
B Cell Precursors

Abstract This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L- 1.

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