scholarly journals A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

1982 ◽  
Vol 208 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Toshihiro Yagura ◽  
Paul G. Walfish
Keyword(s):  
Receptor Binding ◽  
Binding Capacity ◽  
Nuclear Extract ◽  
Affinity Constant ◽  
Lower Sensitivity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Radioimmunoassay Method ◽  
True Values ◽  
Good Agreement

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[125I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.

Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells

1983 ◽  
Vol 61 (7) ◽  
pp. 826-831 ◽  
Author(s):  
T. T. Ilenchuk ◽  
K. G. Davey
Keyword(s):  
Juvenile Hormone ◽  
Binding Capacity ◽  
Follicle Cell ◽  
Follicle Cells ◽  
Follicular Epithelium ◽  
Curvilinear Relationship ◽  
Scatchard Analysis ◽  
Ouabain Binding ◽  
Binding Characteristics ◽  
Brain Microsomes

A comparison has been made of the effects of juvenile hormone (JH) on the binding characteristics for ouabain of microsomes prepared from brain and from cells of the follicular epithelium surrounding previtellogenic or vitellogenic oocytes in Rhodnius. JH has no effect on the binding of ouabain to brain microsomes and decreases the Kd, but does not alter the Bmax for previtellogenic follicle cells. For vitellogenic follicle cells, Scatchard analysis reveals a curvilinear relationship, which is interpreted as indicating that a new population of JH-sensitive ouabain-binding sites develops as the follicle cell enters vitellogenesis. These results are related to earlier data obtained on the effect of JH on ATPase activity, volume changes in isolated follicle cells, and the development of spaces between the cells of the follicular epithelium.

Downregulation of hepatocyte P2-purinoceptor binding capacity after trauma-hemorrhage/resuscitation

1994 ◽  
Vol 266 (6) ◽  
pp. R1804-R1809
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Dissociation Constants ◽  
Binding Capacity ◽  
Isolated Hepatocytes ◽  
Scatchard Analysis ◽  
Midline Laparotomy ◽  
Binding Characteristics ◽  
Shed Blood ◽  
P2 Purinoceptors ◽  
Altered Glucose ◽  
Affinity Receptor

Although P2-purinoceptors play an important role in the regulation of liver metabolism under normal conditions, it is not known if trauma-hemorrhage and resuscitation have any effects on such receptors. To study this, we performed a 5-cm midline laparotomy (i.e., trauma induced) on rats and then bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3x the volume of shed blood with RL over 45 min followed by 2x RL over 95 min. Hepatocytes were isolated at the time of maximum bleedout or at 0, 4, 17, and 27 h after the completion of crystalloid resuscitation. P2-purinoceptor binding characteristics were determined in the isolated hepatocytes by using [alpha-35S]ATP. Scatchard analysis revealed high- and low-affinity components of P2-purinoceptors in hepatocytes from sham-operated as well as hemorrhaged and resuscitated animals. The maximum binding capacity (Bmax) of the high-affinity receptor component decreased at the time of maximum bleedout and at 4, 17, and 27 h after resuscitation. In addition to this, the Bmax of low-affinity receptor components also decreased at 4-27 h after resuscitation. In contrast, the dissociation constants of both receptor components were not altered. Because hemorrhagic shock produces abnormalities in glucose metabolism, the downregulation of hepatocyte P2-purinoceptor Bmax may be responsible for the altered glucose homeostasis under such conditions.

ATP-MgCl2 treatment after trauma-hemorrhage/resuscitation increases hepatocyte P2-purinoceptor binding capacity

1994 ◽  
Vol 266 (6) ◽  
pp. R1810-R1815
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Binding Capacity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Beneficial Effects ◽  
High Affinity ◽  
Receptor Component ◽  
Receptor Dynamics ◽  
Maximum Binding Capacity ◽  
High Affinity Receptor ◽  
Affinity Receptor

Although our studies indicate that P2-purinoceptor binding capacity decreases after hemorrhage and resuscitation, it is not known whether ATP-MgCl2 administration after hemorrhage has any beneficial effects on the receptor dynamics. To study this, we performed laparotomy (i.e., trauma induced) on rats and bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3 times the volume of maximum bleedout with RL over 45 min followed by 2 times RL along with ATP-MgCl2 (50 mumol/kg body wt) over 95 min. Hepatocytes were isolated at 4, 17, and 27 h after resuscitation. P2-purinoceptor binding characteristics were determined by using [alpha-35S]ATP. Scatchard analysis revealed high-affinity and low-affinity receptor components in the hepatocytes isolated from sham-operated or hemorrhaged animals with or without ATP-MgCl2 infusion. ATP-MgCl2 ameliorated and subsequently restored the decreased maximum binding capacity (Bmax) of the high-affinity receptor component and significantly improved Bmax of the low-affinity receptor component. ATP-MgCl2 administration also produced a progressive enhancement in the affinity of the low-affinity receptor component. Thus the beneficial effects of ATP-MgCl2 observed after trauma-hemorrhage and resuscitation may be, in part, due to the restoration of P2-purinoceptor binding capacity and the enhancement of the receptor affinity.

scholarly journals Binding characteristics of antibodies to the TSH receptor

1998 ◽  
Vol 20 (2) ◽  
pp. 233-244 ◽  
Author(s):  
Y Oda ◽  
J Sanders ◽  
S Roberts ◽  
M Maruyama ◽  
R Kato ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Tsh Receptor ◽  
Affinity Constant ◽  
Translation System ◽  
Scatchard Analysis ◽  
Facs Analysis ◽  
Binding Characteristics ◽  
Fab Fragments

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

ATP-MgCl2 administration normalizes macrophage cAMP and beta-adrenergic receptors after hemorrhage and resuscitation

1994 ◽  
Vol 267 (1) ◽  
pp. G52-G58 ◽  
Author(s):  
P. Wang ◽  
S. M. Tait ◽  
Z. F. Ba ◽  
I. H. Chaudry
Keyword(s):  
Receptor Binding ◽  
Kupffer Cells ◽  
Adrenergic Receptors ◽  
Binding Capacity ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Midline Laparotomy ◽  
Binding Characteristics ◽  
Beta Receptor ◽  
Camp Levels

Although ATP-MgCl2 attenuates the release of inflammatory cytokines and restores the defective macrophage (M phi) antigen presentation function after hemorrhage and resuscitation, it is not known whether administration of this agent after hemorrhage affects M phi adenosine 3',5'-cyclic monophosphate (cAMP) levels and beta-adrenergic receptors. To determine this, rats underwent a midline laparotomy (i.e., induction of trauma) and were then bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). Animals were resuscitated with four times the volume of shed blood with RL, during and after which ATP-MgCl2 (50 mumol/kg) or saline was administered over 95 min. At 1.5 h postresuscitation (i.e., 10 min after completion of ATP-MgCl2 infusion), peritoneal M phi and Kupffer cells were isolated, and cAMP levels were measured by radioimmunoassay. beta-Receptor binding characteristics were also determined in isolated Kupffer cells. The results indicate that cAMP levels increased significantly in both peritoneal M phi and Kupffer cells after hemorrhage and resuscitation. Maximum binding capacity (Bmax) of beta-receptors increased in Kupffer cells, suggesting that the elevated cAMP may be due to the increased beta-receptor Bmax under such conditions. ATP-MgCl2 treatment, however, markedly decreased beta-receptor Bmax in Kupffer cells and cAMP in both peritoneal M phi and Kupffer cells, and the values were similar to shams. Thus normalization of M phi cAMP levels and beta-receptor binding capacity by ATP-MgCl2 may contribute to the immunoenhancing effects of this agent observed after trauma-hemorrhage and fluid resuscitation.

Changes in serum somatomedin A and its binding to kidney membranes in growing rats

1981 ◽  
Vol 97 (3) ◽  
pp. 352-357 ◽  
Author(s):  
Naomi Hizuka ◽  
Kazue Takano ◽  
Kazuo Shizume ◽  
Yoko Hasumi
Keyword(s):  
Binding Capacity ◽  
Inverse Correlation ◽  
Close Correlation ◽  
Serum Levels ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Receptor Sensitivity ◽  
Growing Rats ◽  
The Mean ◽  
Clear Change

Abstract. Changes in serum somatomedin A levels and [125I]somatomedin A binding to membrane fractions from kidney were studied in rats 1–80 days of age. The mean level of serum somatomedin A was 0.80 U/ml at birth and increased with age; at 80 days the mean level was 7.41 ± 0.67 U/ml. There was a close correlation between serum levels of somatomedin A and body weight. Labelled somatomedin A binding to membrane fractions from kidney was highest at birth and decreased with age up to 50 days. In Scatchard analysis of the data the affinity constant did not show a clear change with age, but the binding capacity decreased with age up to 30 days. An inverse correlation was observed between serum somatomedin A levels and labelled somatomedin A binding to membrane fractions from kidney. Compared to changes in circulating somatomedin A, the change in tissue binding was modest. This observation suggests that other circulating growth factors not measured by this radioreceptor assay or altered post-receptor sensitivity to somatomedins may be involved in growth.

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

1990 ◽  
Vol 68 (3) ◽  
pp. 616-621 ◽  
Author(s):  
Shinko Takagi ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Specific Activity ◽  
Binding Capacity ◽  
High Specific Activity ◽  
Relative Mass ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Therapeutic Effects ◽  
Inhibitory Effects

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass ≈ 50 000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 μCi/μg; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 × 109 to 1.74 × 109 M−1 for PTU and 1.79 × 109 M−1 for MMI), while they both decreased (p < 0.02) maximal binding capacity (from 0.36 ± 0.02 to 0.19 ± 0.02 pmol/mg protein for MMI and 0.17 ± 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with ≈25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats. Pretreatment of CNR for 1 h with 5 mM methyl methanethiosulfonate (an oxidant of thiol groups) abolished the inhibitory effects of PTU, MMI, or arsenite, but was not inhibitory in itself. From these studies it is concluded that (i) MMI and PTU could exert at least part of their therapeutic effects by inhibiting specific binding of L-T3 to its hepatic nuclear receptor; (ii) these inhibitory effects of MMI and PTU are likely due to an interaction with cysteine residues (some of which are not in a dithiol configuration) that are essential for T3 binding to its receptor; and (iii) binding of T3 is not inhibited by oxidation of receptor thiols to methyl dithiol groups.Key words: nuclear triiodothyronine (T3) receptor, methimazole, propylthiouracil, Scatchard analysis.

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors

1989 ◽  
Vol 67 (8) ◽  
pp. 477-480 ◽  
Author(s):  
Shinko Takagi ◽  
Ganesh B. Bhat ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Glutathione Reductase ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Thioredoxin Reductase ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Physiological Effects ◽  
Thioredoxin System

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 ± 0.1 × 109 M−1 MBC = 1.7 ± 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.Key words: nuclear triiodothyronine (T3) receptor, thioredoxin, glutaredoxin, Scatchard analysis.

Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 150 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M J Pesek ◽  
M A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
High Affinity ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
C Terminus ◽  
Liver Membranes

Abstract Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding. Journal of Endocrinology (1996) 150, 179–186

In vivo Binding of Nimodipine in the Brain: II. Binding Kinetics in Focal Cerebral Ischemia

1991 ◽  
Vol 11 (5) ◽  
pp. 771-778 ◽  
Author(s):  
Matthew J. Hogan ◽  
Albert Gjedde ◽  
Antoine M. Hakim
Keyword(s):  
Cerebral Ischemia ◽  
Calcium Channel ◽  
Focal Cerebral Ischemia ◽  
Binding Capacity ◽  
Affinity Constant ◽  
Binding Characteristics ◽  
In Vivo Binding ◽  
Ischemic Tissue ◽  
The Brain

We report the binding characteristics of [3H]nimodipine to normal and ischemic brain in vivo. We used the 1,4-dihydropyridine, nimodipine, to label the L-type voltage-sensitive calcium channel in focal cerebral ischemia after occlusion of both the middle cerebral and ipsilateral common carotid arteries in rats. Varying concentrations of [3H]nimodipine were infused 3.5 h after the onset of ischemia and circulated for 30 min before the brain was obtained for autoradiography and determination of regional nimodipine content. In separate sets of experiments, the metabolites of nimodipine were determined and the conditions for equilibrium of nimodipine distribution were established. Increased nimodipine uptake was observed in ischemic regions. This increased binding was saturable and specific with an affinity constant, KD, of 0.45 n M and a maximal regional binding capacity, Bmax, ranging from 3.1 to 10.9 pmol/g. Only binding to ischemic tissue was specific and saturable whereas that in nonischemic tissue was nonspecific. In vivo binding of nimodipine may be used to identify cell membrane depolarization and calcium channel activation in focal cerebral ischemia.

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