[3H]norharman ([3H]?-carboline) binds reversibly and with high affinity to a specific binding site in rat liver

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.

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Characterization of a high-affinity and specific binding site for Gas6

FEBS Letters ◽

10.1016/0014-5793(96)00394-8 ◽

1996 ◽

Vol 387 (1) ◽

pp. 75-77 ◽

Cited By ~ 9

Author(s):

Toru Nakano ◽

Junji Kishino ◽

Hitoshi Arita

Keyword(s):

Binding Site ◽

Specific Binding ◽

High Affinity

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Characterization of galanin receptor in canine small intestinal circular muscle synaptosomes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.1.g106 ◽

1994 ◽

Vol 266 (1) ◽

pp. G106-G112 ◽

Cited By ~ 4

Author(s):

C. K. Chen ◽

T. J. McDonald ◽

E. E. Daniel

Keyword(s):

Small Intestine ◽

Binding Site ◽

G Protein ◽

Binding Capacity ◽

Specific Binding ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Circular Muscle ◽

Galanin Receptor ◽

High Affinity

We used 125I-galanin (porcine) as ligand to study the galanin receptors in circular muscle and deep muscular plexus from canine small intestine. Specific binding sites were found in both nerve and muscle membranes. On synaptosomal membranes, the equilibrium binding study showed a high-affinity (dissociation constant, Kd = 1.1 +/- 0.13 nM; maximum binding capacity, Bmax = 244 +/- 2.1 fmol/mg) binding site. The specific binding of 125I-galanin to nerve membrane was inhibited by galanin or NH2-terminal galanin fragments but not by the COOH-terminal fragment. Computer analysis suggested a two-site model (inhibitor constants, Ki1 = 0.02 +/- 0.005 nM and Ki2 = 1.05 +/- 0.3 nM) for competition by galanin-(1-29). Kinetic and competition studies using guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin (PTX) suggested that the high-affinity binding site involved a PTX-sensitive G protein which acted to slow dissociation of bound galanin from the receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the galanin receptor complex revealed a radioactive band at 50 kDa. We conclude that, in canine small intestine, galanin may act as an inhibitory neuromodulator by a PTX-sensitive G protein-coupled interaction of galanin and its specific receptor on enteric nerve synaptosomes

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Cross-reactivity of amylin with calcitonin-gene-related peptide binding sites in rat liver and skeletal muscle membranes

Biochemical Journal ◽

10.1042/bj2770139 ◽

1991 ◽

Vol 277 (1) ◽

pp. 139-143 ◽

Cited By ~ 45

Author(s):

A Chantry ◽

B Leighton ◽

A J Day

Keyword(s):

Skeletal Muscle ◽

Binding Site ◽

Rat Liver ◽

Specific Binding ◽

Calcitonin Gene Related Peptide ◽

Cross Reactivity ◽

Scatchard Analysis ◽

Human Calcitonin ◽

Related Peptide ◽

Calcitonin Gene

This study examines whether the high degree of sequence identity between amylin and calcitonin-gene-related peptide (CGRP) is reflected in their cross-reactivity at the level of membrane receptor binding. Rat liver plasma membranes contain a specific saturable binding site for 125I-labelled human CGRP-1. Binding reached equilibrium within 30 min and was rapidly reversed by re-incubating membranes in the presence of 1 microM human CGRP. In addition, the presence of 50 mM- or 500 mM-NaCl lowered specific binding by 30% and 77% respectively. Scatchard analysis was consistent with a single high-affinity site with a dissociation constant (Kd) of 0.125 nM and binding capacity (Bmax.) of 580 fmol/mg of membrane protein. Specific binding of 125I-labelled human CGRP-1 to both liver and skeletal muscle membranes was inhibited by human CGRP-1 [IC50 (concn. causing half-maximal inhibition of binding) 0.1-0.3 nM], and rat amylin (IC50 10 nM), but not by human calcitonin. Covalent cross-linking of 125I-CGRP to its binding site in rat skeletal muscle and liver membranes resulted in labelling of a major species of about 70 kDa under reducing conditions and about 55 kDa under alkylating conditions, as visualized on SDS/PAGE. These radiolabelled species were absent in the presence of CGRP or amylin at 1 microM. These results are indicative of a common binding site for both CGRP and amylin in liver and skeletal muscle, and it is suggested that both peptides mediate their actions through the same effector system. The normal physiological importance and the relevance to the pathology of type 2 diabetes of these data are discussed.

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Studies on the nature of the high-affinity trialkyltin binding site of rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2020163 ◽

1982 ◽

Vol 202 (1) ◽

pp. 163-169 ◽

Cited By ~ 20

Author(s):

A P Dawson ◽

B G Farrow ◽

M J Selwyn

Keyword(s):

Binding Site ◽

Rat Liver ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Affinity Binding ◽

Mitochondrial Atpase ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site ◽

Triphenyltin Chloride

1. The proteolipid fraction isolated from rat liver mitochondria pretreated with [3H]triphenyltin chloride is enriched in triphenyltin compared with the original mitochondria. 2. Part of this [3H]triphenyltin is eluted with a protein of Mr 5000-6000 on Sephadex LH20 chromatography. 2. Mössbauer spectra of the proteolipid fraction treated with 119Sn-enriched triethyltin chloride show a doublet which corresponds closely with that assigned previously [Farrow & Dawson (1978) Eur. J. Biochem. 86. 85-95] to the absorption of triethyltin bound to the high-affinity binding site of the mitochondrial ATPase.

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Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum

Biochemical Journal ◽

10.1042/bj2740861 ◽

1991 ◽

Vol 274 (3) ◽

pp. 861-867 ◽

Cited By ~ 48

Author(s):

R A J Challiss ◽

A L Willcocks ◽

B Mulloy ◽

B V L Potter ◽

S R Nahorski

Keyword(s):

Binding Site ◽

Binding Sites ◽

Radioligand Binding ◽

Specific Binding ◽

Binding Activity ◽

Inositol Polyphosphate ◽

Homogeneous Population ◽

Site Model ◽

High Affinity ◽

Pentosan Polysulphate

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude ‘P2’ cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

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

Biochemical Journal ◽

10.1042/bj2250473 ◽

1985 ◽

Vol 225 (2) ◽

pp. 473-479 ◽

Cited By ~ 58

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.

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High-and Low-Affinity Binding of Photosystem II Herbicides to Isolated Thylakoid Membranes and Intact Algal Cells

Zeitschrift für Naturforschung C ◽

10.1515/znc-1982-7-811 ◽

1982 ◽

Vol 37 (7-8) ◽

pp. 620-631 ◽

Cited By ~ 25

Author(s):

Henrik Laasch ◽

Klaus Pfister ◽

Wolfgang Urbach

Keyword(s):

Photosystem Ii ◽

Binding Site ◽

Spinacia Oleracea ◽

Specific Binding ◽

Affinity Binding ◽

Concentration Effects ◽

Intact Cells ◽

High Affinity Binding ◽

High Affinity ◽

Receptor Sites

Abstract High- and low-affinity binding of photosystem II herbicides to isolated thylakoids of Spinacia oleracea and to intact cells of the unicellular green alga Ankistrodesmus braunii were investigated. Complete mutual displacement of bound diuron-type herbicides (e.g. diuron, atrazine, terbutryn) by either diuron- or phenol-type herbicides (e.g. ioxynil, dinoseb) in thylakoids as well as in intact algal cells was found for herbicide concentrations (< 4 nmol bound herbicide/mg Chl) which gave almost saturated high-affinity binding. This demonstrates a high degree of specific binding of these herbicides towards their receptor sites even in intact algal cells. In contrast, phenol-type herbicides are largely unspecifically bound in algal cells. The mechanism of binding of all photosystem II herbicides at the high-affinity (specific) binding site was found to be competitive. Within the group of diuron-type and of phenol-type herbicides as well as between these two groups, graphical and quantitative analysis of the Lineweaver- Burk plot and of the Dixon plot indicated competitive binding. From this a common binding site for both types of herbicides was concluded. The involvement of two different herbicide binding- proteins is discussed. Low-affinity (unspecific) binding was found to be irreversible in contrast to the easily reversible high-affinity binding. Irreversibility was indicated by a lack of displacement. It is proposed that low-affinity binding represents either a partitioning of the herbicides into the lipophilic parts of the membranes or an attachment to distinct receptor sites. Unspecifically bound herbicides might be responsible for several high concentration effects of the photosystem II herbicides, which are described in the literature. Evidences for the possible existence of a second binding site of these herbicides are presented.

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Somatotrophic receptors in hepatic tissue of the developing male pig

Journal of Endocrinology ◽

10.1677/joe.0.1230025 ◽

1989 ◽

Vol 123 (1) ◽

pp. 25-31 ◽

Cited By ~ 36

Author(s):

B. H. Breier ◽

P. D. Gluckman ◽

H. T. Blair ◽

S. N. McCutcheon

Keyword(s):

Binding Site ◽

Specific Binding ◽

Plasma Concentrations ◽

Age Groups ◽

Affinity Binding ◽

Lower Affinity ◽

High Affinity ◽

Igf I ◽

High Affinity Binding Site ◽

Scatchard Plots

ABSTRACT The development of hepatic somatotrophic receptors and plasma concentrations of insulin-like growth factor-I (IGF-I) were investigated at five different ages (2, 20, 35, 105 and 165 days) in four male pigs per group. The specific binding of 125I-labelled porcine GH (pGH) to hepatic somatotrophic membranes was very low at 2 days of age (0·53±0·12%), and increased progressively (P <0·01) with advancing age to 3·60 ± 0·95% at 165 days of age. Specific binding of 125I-labelled bovine GH (bGH) to the same membrane preparations was markedly higher than binding of 125I-labelled pGH; it also showed a distinct developmental increase (P <0·01) with age from 4·4 ± 0·55% at 2 days of age to 24·0±1·90% at 165 days of age. Plasma concentrations of IGF-I increased significantly (P <0·01) from 79 ± 14·0 μg/l at 2 days of age to 610 ± 64·0 μg/l at 165 days of age. Non-linear regression analysis of the competitive binding data using bGH as labelled and unlabelled ligands showed linear Scatchard plots in the three youngest age groups, with an association constant (Ka) of approximately 3·5 litres/nmol. Curvilinear Scatchard plots were observed in the two oldest age groups. The Ka for the higher affinity binding site (approximately 5·0 litres/nmol) was very similar to that for the sole site observed in the younger animals. The Ka of the lower affinity binding site was approximately 0·35 litres/nmol. There was a significant (P <0·01) developmental increase in the capacity of the higher affinity binding site from 12 ± 4·6 pmol/100 mg liver at 2 days of age to 91 ± 23·0 pmol/100 mg liver at 165 days of age. These studies demonstrate heterogeneity of somatotrophic hepatic membranes in the pig and show that low concentrations of a high affinity binding site are already present in newborn pigs. A considerable developmental increase was observed in the capacity of this binding site which correlated significantly (r = 0·82, P <0·01, n = 20) with plasma concentrations of IGF-I. The role of the lower affinity binding site which was observed in addition to the high affinity binding site in older pigs is less clear. The data from the present study support the hypothesis that the postnatal rise in plasma concentrations of IGF-I is associated with the developmental increase of the capacity of the high affinity somatotrophic receptors. Journal of Endocrinology (1989) 123, 25–31

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Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver

Biochemical Journal ◽

10.1042/bj2020225 ◽

1982 ◽

Vol 202 (1) ◽

pp. 225-230 ◽

Cited By ~ 10

Author(s):

Y A Lefebvre ◽

S J Morante

Keyword(s):

Heat Treatment ◽

Nuclear Envelope ◽

Binding Site ◽

Rat Liver ◽

Binding Sites ◽

Specific Binding ◽

Male Rat ◽

Scatchard Analysis ◽

Female Rat ◽

Similar Class

Intact nuclear ‘ghosts’ containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.

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