New aspects of the physiological significance of LRH receptors of pituitary plasma membranes

1981 ◽  
Vol 96 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Herbert Kuhl ◽  
Rudolf Baumann
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Functional Relationship ◽  
Metabolic Pattern ◽  
High Affinity ◽  
Highly Active ◽  
Rat Pituitary ◽  
Intracellular Compartments ◽  
Triton X ◽  
Two Peaks

Abstract. It has been found that l-cystine-bis-(4-nitroanilide), a synthetic peptidase substrate, competes with [125I]LRH for specific high affinity LRH receptors on isolated rat pituitary plasma membranes as effectively as a highly active LRH analogue. The pattern of [125I]LRH degradation by isolated pituitary plasma membranes was similar to that effected by pituitary and hypothalamic enzyme extracts. The enzyme activity seemed to accumulate in the supernatant of the membrane suspension after being released from the membrane structure. Plasma membrane supernatant and pituitary and hypothalamic enzyme extracts were chromatographed together with labelled LRH on Sephadex G 200. This resulted in protein binding patterns with one peak at the fractions representing a molecular weight of 600 000 to 650 000 and another one of approximately 80 000, the first probably being an aggregate of the latter. After solubilization of the plasma membranes by Triton X-100, there was no evidence for another LRH receptor. There were also two peaks of [125I]LRH degrading activity (with identical metabolic pattern) and two peaks of arylamidase activity, which were, however, not identical with the binding peaks. Both the arylamidase and the LRH binding-pattern had been demonstrated to be temperature- and hormonedependent, and seemed to be functionally related. The results indicate that there is a functional relationship between LRH binding-sites on plasma membranes and LRH degrading enzymes. This implies that the search for the 'true' LRH receptor has to be directed towards intracellular compartments of the pituitary gonadotrophs.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

Anti-TSH receptor antibodies in Graves' disease modulate the kinetic behaviour of autologous thyroid TSH receptors. Evidence of the IgG-induced cross-linkage of autologous TSH receptors

1984 ◽  
Vol 105 (3) ◽  
pp. 330-340 ◽  
Author(s):  
Tjerk W. A. de Bruin ◽  
Daan van der Heide ◽  
Maria C. Krol
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Graves Disease ◽  
Kinetic Behaviour ◽  
High Affinity ◽  
Cross Linkage ◽  
Receptor Antibodies ◽  
Tsh Receptor Antibodies

Abstract. The effect of the anti-TSH receptor antibodies present in the sera of 8 patients with Graves' disease on the affinity constant (Ka) and the number (R) of TSH receptors in autologous human thyroid plasma membranes was investigated. Kinetic analysis of [125I]bTSH binding to human thyroid plasma membranes in the presence of autologous Graves' and normal gammaglobulins was carried out by means of a computer fitting programme. Analysis of the TSH-TSH receptor interaction in the presence of TSH alone yielded curvilinear Scatchard plots, indicating the existence of two independent classes of binding sites (high affinity Ka: 8.5 ± 4.8 × 108 m−1; low affinity Ka: 5.3 ± 2.7 × 106 m−1). Similarly the Scatchard plot for this interaction in the presence of normal gammaglobulins is also curvilinear. Linear Scatchard plots, indicating the existence of only one class of high affinity TSH binding sites (Ka: 3.5 ± 1.8 × 108 m−1), were obtained for both autologous gammaglobulins and pure IgG from 8 patients with Graves' disease. The number of high affinity TSH binding sites in the presence of Graves' gammaglobulins had increased on the average by a factor 3.76 ± 0.74 (sd) with respect to the number found in the presence of normal gammaglobulins. This marked change in the kinetic behaviour of the TSH binding sites provided evidence that there is a direct interaction between anti-TSH receptor antibodies and autologous TSH receptors. Divalency of Graves' IgG or linkage of Fab fragments by anti-Fab antiserum proved to be necessary to produce this specific change in the kinetic behaviour of TSH binding sites. Graves' IgG monovalent Fab and Fc fragments had no effect. We suggest that the mechanism by which anti-TSH receptor antibodies in Graves' disease mimick the biological action of TSH is the IgG-induced cross-linkage of TSH receptors.

High affinity binding sites for somatostatin to rat pituitary

1982 ◽  
Vol 105 (4) ◽  
pp. 1538-1545 ◽  
Author(s):  
J.-C. Reubi ◽  
M. Perrin ◽  
J. Rivier ◽  
W. Vale
Keyword(s):  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Rat Pituitary

scholarly journals Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

1977 ◽  
Author(s):  
J. Lips ◽  
J. J. Sixma
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
High Affinity ◽  
Absolute Requirement ◽  
Millipore Filtration

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP

1986 ◽  
Vol 64 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Denis LeBel ◽  
Marlyne Beattie
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Photoaffinity Labeling ◽  
Competitive Inhibitor ◽  
Atp Binding ◽  
Zymogen Granule ◽  
Atp Diphosphohydrolase ◽  
High Affinity ◽  
Granule Membrane ◽  
Triton X

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

Fungal Peptide Elicitors: Signals Mediating Pathogen Recognition in Plants

1998 ◽  
Vol 53 (3-4) ◽  
pp. 141-150 ◽  
Author(s):  
Thorsten Nürnberger ◽  
Dirk Nennstiel
Keyword(s):  
Plant Defense ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Pathogen Resistance ◽  
Microbial Pathogens ◽  
Functional Link ◽  
High Affinity ◽  
Highly Sensitive ◽  
Recognition Systems

Abstract Highly sensitive and specific recognition systems for microbial pathogens are essential for disease resistance in plants. Proteinaceous elicitors activating plant pathogen defense have been identified in numerous antagonistic plant/fungus interactions. Precisely defined signal structures required for elicitor-mediated activation of plant defense are indicative of the involvement of receptors in elicitor perception and subsequent signal generation. Use of pure elicitor preparations has helped to establish a functional link between binding of elicitors to high-affinity binding sites in plant plasma membranes and activation of plant defense. Thus elicitor binding sites appear to function as physiological receptors. Currently, isolation and molecular characterization of elicitor receptors is under way. Transfer of new recognition specificities into plants is supposed to be a key strategy for engineering pathogen resistance in economically important crops.

scholarly journals The rat liver vasoactive intestinal peptide binding site. Molecular characterization by covalent cross-linking and evidence for differences from the intestinal receptor

1985 ◽  
Vol 225 (2) ◽  
pp. 473-479 ◽  
Author(s):  
A Couvineau ◽  
M Laburthe
Keyword(s):  
Binding Site ◽  
Vasoactive Intestinal Peptide ◽  
Rat Liver ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
High Affinity ◽  
Cross Linker ◽  
A Minor

To identify the molecular components of the vasoactive intestinal peptide (VIP) binding sites in the liver, 125I-labelled VIP was covalently linked to liver membranes by using the cleavable cross-linker dithiobis(succinimidylpropionate). Purified rat liver plasma membranes were incubated with 125I-VIP, washed and treated with 1 mM-cross-linker. Polyacrylamide-gel electrophoresis of membrane proteins followed by autoradiography revealed a major 125I-VIP-protein complex of Mr 51 000. A minor Mr 89 000 complex was also observed. An identical pattern of protein labelling was obtained using crude membranes from rat liver. Labelling of the Mr 51 000 and 89 000 species was specific in that it could be abolished by native VIP, but was unaffected by 1 microM-glucagon and cholecystokinin octapeptide. Densitometric scanning of autoradiographs indicated that the labelling of the two species was abolished by similar low VIP concentrations (0.1-100 nM). It was also reduced by two VIP agonists, peptide histidine isoleucine amide and secretin, with a potency that is 1/7 and 1/200 that of native VIP, respectively. The guanine nucleotide GTP in the concentration range between 10(-7) and 10(-3) M reduces the labelling of the major Mr 51 000 protein and that of the minor Mr 89 000 protein, but with a slightly higher potency. Assuming one molecule of 125I-VIP was bound per molecule of protein, a major Mr 48 000 protein and a minor Mr 86 000 protein were identified as components of the high-affinity VIP binding sites in liver. This contrasts markedly with the pattern of labelling of rat intestinal epithelial membranes, where a Mr 73 000 protein was identified as a high-affinity VIP receptor and a Mr 33 000 protein as a low-affinity VIP binding site [Laburthe, Bréant & Rouyer-Fessard (1984) Eur. J. Biochem. 139, 181-187], suggesting structural differences between VIP binding sites in rat liver and intestinal epithelium.

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland.: Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)51[Tyr(Me)2, Thr4,Tyr-NH29]OVT

1989 ◽  
Vol 67 (2-3) ◽  
pp. 152-162 ◽  
Author(s):  
Melvyn S. Soloff ◽  
Mats A. Fernström ◽  
Martha J. Fernström
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Inositol Phosphate ◽  
Mammary Tissue ◽  
Selective Antagonist ◽  
High Affinity ◽  
Oxytocin Receptors ◽  
Rat Mammary Gland ◽  
Stimulation Of

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)51[Tyr(Me)2,Thr4,Tyr-NH29]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)51[Tyr(Me)2,Thr4,Tyr-NH29]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding. The high affinity, specificity, and specific activity of the iodinated antagonist should make it very useful in further studies to discriminate between oxytocin and AVP receptors, demonstrate oxytocin receptors with small amounts of samples, perform autoradiographic studies with short-term exposures, and to purify oxytocin receptors.Key words: oxytocin, vasopressin, receptor, mammary gland, antagonist.

scholarly journals Guanosine nucleotides regulate hormone binding of insulin receptors

1992 ◽  
Vol 281 (3) ◽  
pp. 735-743 ◽  
Author(s):  
E R Mortensen ◽  
J Drachman ◽  
G Guidotti
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Temperature Dependent ◽  
High Affinity ◽  
Mild Reduction ◽  
Adipocyte Plasma Membranes

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

Sign in / Sign up

Export Citation Format

Share Document