Binding of [14,15-3H]14,15-dihydroforskolin to rat liver membranes: comparison with the stimulatory effect of forskolin on adenylate cyclase

1987 ◽  
Vol 65 (5) ◽  
pp. 803-809 ◽  
Author(s):  
Kurt Schmidt ◽  
Hans P. Baer ◽  
Azim Shariff ◽  
William A. Ayer ◽  
Lois Browne
Keyword(s):  
Adenylate Cyclase ◽  
Rat Liver ◽  
Binding Sites ◽  
Binding Capacity ◽  
Protein A ◽  
High Specificity ◽  
Binding Studies ◽  
Liver Membranes ◽  
High Concentrations ◽  
Equilibrium Dissociation

The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 μM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 μM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure–activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (KI = 0.2 μM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-β-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations, [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin.

Analysis of the lipoprotein binding site of rat liver membranes

1994 ◽  
Vol 72 (3-4) ◽  
pp. 132-142 ◽  
Author(s):  
Lynda Adam ◽  
Louise Brissette
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
New Information ◽  
Liver Membrane ◽  
Liver Membranes ◽  
Bona Fide ◽  
Lipoprotein Binding

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.Key words: lipoprotein, receptor, binding, metabolism.

Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial α-adrenoceptors in the ewe

1990 ◽  
Vol 127 (3) ◽  
pp. 471-479 ◽  
Author(s):  
A. Vass-Lopez ◽  
R. Garcia-Villar ◽  
M. Lafontan ◽  
P. L. Toutain
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Steroid Hormone ◽  
Physiological Status ◽  
Equilibrium Dissociation Constant ◽  
Binding Studies ◽  
Saturation Binding ◽  
Adrenergic Antagonists ◽  
Equilibrium Dissociation

ABSTRACT The adrenergic antagonists [3H]prazosin and [3H] rauwolscine were used to identify α1- and α2-adrenoceptors respectively in the ovine myometrium. Ewes were allocated to four groups according to steroid hormone treatments or physiological status, namely ovariectomized ewes either as untreated controls, treated with oestradiol-17β or progestagen plus oestradiol-17β, and pregnant ewes at mid-gestation. Binding of both [3H]prazosin and [3H]rauwolscine to membrane preparations from the ovine myometrium was saturable, of high affinity and rapidly reversed by phentolamine (10 μmol/l). Based on the relative order of potency of selected adrenergic agonists and antagonists, the myometrial binding sites labelled by [3H]prazosin and [3H]rauwolscine were characterized as α1- and α2adrenoceptors respectively. Saturation binding studies with [3H]prazosin showed that the number of α1adrenoceptors was low (maximal binding capacity, Bmax, between 19 and 24 fmol/mg protein) and there were no noticeable differences between the animal groups. Moreover, the equilibrium dissociation constant (Kd) did not vary significantly between groups (Kd between 0·10 and 0·17 nmol/l). In contrast, saturation binding studies with [3H]rauwolscine revealed the presence of a high number of α2-adrenoceptors. Values of Bmax were far higher in the pregnant ewes (1096±241 fmol/mg protein; means ± s.d.) than in any of the non-pregnant ovariectomized ewes. For these latter groups, the highest Bmax values were found in the group treated with both progestagen and oestrogen (382±77 fmol/mg protein) compared with treatment with oestrogen alone (101±8 fmol/mg protein) or with controls (82±12 fmol/mg protein). The results of the present study, especially those obtained under a high-progesterone environment (e.g. pregnancy), strongly suggest a role of steroid hormones in the control of the number of α2-adrenoceptors in the ovine myometrium. In contrast, they did not yield any supporting data for a similar role on myometrial α1-adrenoceptors in the ewe. Journal of Endocrinology (1990) 127, 471–479

Insulin Binding Sites Induced in the Tetrahymena by Rat Liver Receptor Antibody

1984 ◽  
Vol 39 (1-2) ◽  
pp. 183-185 ◽  
Author(s):  
G. Csaba ◽  
P. Kovács ◽  
Ágnes Inczefi-Gonda
Keyword(s):  
Rat Liver ◽  
Concanavalin A ◽  
Binding Sites ◽  
Binding Capacity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Receptor Antibody ◽  
Receptor Antibodies

Abstract Tetrahvmena cells treated with purified rabbit anti­ bodies to rat hepatocellular membrane exhibited a consider­ able increase in binding capacity on reexposure to the antibody 24 h later. Insulin binding was similarly enhanced by preexposure to the antibody, and vice versa, preex­ posure to insulin enhanced the later binding of rat liver receptor antibodies. This suggests that (1) the Tetrahymena and the rat possess similar insulin receptors, and (2) the receptor antibody is also able to induce imprinting for itself as well as for insulin. Concanavalin-A, noted for binding overlap with insulin, failed to induce imprinting either for insulin or for antibodies to receptors, whereas the latter did induce imprinting for Concanavalin-A.

Pituitary regulation of human growth hormone binding sites in rat liver membranes

Metabolism ◽  
1976 ◽  
Vol 25 (3) ◽  
pp. 341-353 ◽  
Author(s):  
A.C. Herington ◽  
L.S. Phillips ◽  
W.H. Daughaday
Keyword(s):  
Growth Hormone ◽  
Rat Liver ◽  
Human Growth Hormone ◽  
Binding Sites ◽  
Human Growth ◽  
Hormone Binding ◽  
Liver Membranes

scholarly journals The association in vitro of polyribosomes with ribonuclease-treated derivatives of hepatic rough endoplasmic reticulum. Characteristics of the membrane binding sites and factors influencing association

1971 ◽  
Vol 125 (1) ◽  
pp. 67-79 ◽  
Author(s):  
T. K. Shires ◽  
L. Narurkar ◽  
H. C. Pitot
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Binding Capacity ◽  
Trypsin Treatment ◽  
Partial Removal ◽  
Adult Rat ◽  
Microsomal Membranes ◽  
Derivatives Of ◽  
Membrane Binding Sites

1. Pancreatic ribonuclease in dilute EDTA has been shown to condition rough-microsomal membranes from adult rat liver to accept exogenously added rat liver polyribosomes in vitro at 0–4°C. Treated smooth membranes would not significantly interact with polyribosomes. 2. The conditioning process decreased the membrane RNA content and removed polyribosomes from vesicle surfaces as viewed electron-microscopically. 3. Binding to these conditioned membranes was shown to be uninfluenced by changes of temperature (0–37°C) and pH (6.9–7.8) or the presence of cell sap, but was inhibited by increasing the concentration of potassium chloride. 4. Possession of a polyribosome-binding capacity by conditioned rough membranes was not dependent on adventitious materials that could be dislodged by high ionic strengths. 5. Trypsin treatment under mild conditions destroyed the binding capacity of ribonuclease-conditioned rough membranes. 6. A 2–10S residual RNA was recovered from ribonuclease-conditioned membranes, but its partial removal had no effect on the capacity of membranes to accept polyribosomes. However, some role for this residual RNA in attaching polyribosomes could not be discounted. 7. Evidence is considered that polyribosome-binding sites are intrinsic features of conditioned membranes isolated from rough-microsomal fractions, and that long-range ionic bonding is a primary factor in polyribosome interaction with these binding sites.

scholarly journals The Sulphide-Binding Protein in the Blood of the Vestimentiferan Tube-Worm, Riftia Pachyptila, is the Extracellular Haemoglobin

1987 ◽  
Vol 128 (1) ◽  
pp. 139-158 ◽  
Author(s):  
ALISSA J. ARP ◽  
JAMES J. CHILDRESS ◽  
RUSSELL D. VETTER
Keyword(s):  
Hydrothermal Vent ◽  
Binding Sites ◽  
Binding Capacity ◽  
Binding Protein ◽  
Coelomic Fluid ◽  
Oxygen Binding ◽  
Riftia Pachyptila ◽  
Dissociation Product ◽  
High Concentrations ◽  
Vent Tube

The sulphide-binding protein that occurs in high concentrations in the vascular blood and coelomic fluid of the hydrothermal vent tube-worm Riftia pachyptila Jones is the haemoglobin. Sulphide binding does not occur at the oxygen-binding sites of the haem, but may occur via thiol-disulphide exchange at the interchain disulphide bridges on the macromolecule. We have confirmed the report that vascular blood is heterogeneous for two haemoglobins (FI and FII) that are different in Mr, but we conclude that the coelomic fluid is homogeneous for the lower Mr haemoglobin FII, in the intact, living animal. These two haemoglobins occur naturally in the living animals, and FII is not a dissociation product of the higher Mr FI. The sulphide-binding capacities of the two haemoglobin species differ by about a factor of two. Consequently, the vascular blood and the coelomic fluid also have different sulphide-binding capacities. These differences in sulphide-binding capacity may have important ramifications for the physiology of this unusual animal.

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.

THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE

1977 ◽  
Vol 74 (2) ◽  
pp. 323-334 ◽  
Author(s):  
A. C. HERINGTON ◽  
N. M. VEITH
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Sites ◽  
Specific Binding ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Membrane Function ◽  
Liver Membranes

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]α-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 °C, binding reached a steady-state after 2·5 h and had a half-life of dissociation of 2–3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26·74 ± 3·73 fmol/106 cells and a binding affinity of 1·24 × 109 ± 0·17 × 109 (s.e.m.) l/mol (n = 10). There was a significant sex difference in binding (female > male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.

Characterization of the binding sites for [3H]glibenclamide in rat liver membranes

1995 ◽  
Vol 284 (1-2) ◽  
pp. 77-82 ◽  
Author(s):  
Yasushi Inoue ◽  
Masahiro Emoto ◽  
Hiroshi Inoue ◽  
Kohei Kaku ◽  
Toshio Kaneko
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Liver Membranes
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