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.

Structural characterization of PACAP receptors on rat liver plasma membranes

1993 ◽  
Vol 265 (5) ◽  
pp. G811-G818
Author(s):  
T. D. Nguyen ◽  
G. G. Heintz ◽  
M. S. Wolfe
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Receptors ◽  
Receptor Complexes ◽  
High Affinity ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Pacap Receptors

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and PACAP-27 are recently characterized hypothalamic peptides with marked homology with vasoactive intestinal peptide (VIP), which are concentrated in the innervation of the digestive tract. We now report that, on rat liver plasma membranes, PACAP interacts with at least two types of receptors: receptors demonstrating equally high affinity for PACAP and VIP and receptors with high affinity for PACAP but low affinity for VIP. In contrast, on rat intestinal epithelial cell laterobasal membranes, only receptors with high affinities for PACAP and VIP were observed. After 125I-labeled VIP or 125I-labeled PACAP-27 was cross-linked to the liver plasma membrane receptors with the use of either disuccinimidosuberate or disuccinimido dithiobis(propionate), analysis of the resulting ligand-receptor complexes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the structures of the VIP and PACAP receptors were similar: both ligand-receptor complexes displayed two radioactive bands with relative molecular weights of 80,000 and 56,000 under reducing conditions and of 75,000 and 53,000 under nonreducing conditions. These findings suggest that the receptors for the PACAP peptides and VIP are closely related, reflecting the marked homology between these peptides. The presence of receptors specific for PACAP on rat liver plasma membranes should stimulate further studies of the interaction between PACAP and the liver.

Hepatic Binding Sites for Angiotensin II in the Rat

1979 ◽  
Vol 56 (1) ◽  
pp. 33-40 ◽  
Author(s):  
J. J. Lafontaine ◽  
M. P. Nivez ◽  
R. Ardaillou
Keyword(s):  
Angiotensin Ii ◽  
Rat Liver ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Liver Membranes ◽  
Clear Increase

1. 125I-labelled (Asn1, Val5)-angiotensin II (125I-labelled AII) incubated with purified rat liver membranes was degraded with time, as estimated by three techniques: binding to an excess of specific antibody, polyacrylamide-gel electrophoresis and rebinding to fresh membranes. Degradation was inhibited in the presence of an excess of β1–24-corticotrophin but still very marked. 2. 125I-labelled AII became bound to purified rat liver membranes. Association and dissociation rates were slow. Binding was competitively inhibited by (Asn1, Val5)-AII, (Asp1, Ile5)-AII and (Des, Asn1, Ile5)-AII. Apparent KD was approximately 0·1 nmol/l. 3. Bound hormone was also partly degraded independently of time. 4. Angiotensinases inhibitors had different effects on 125I-labelled AII binding. A clear increase was observed in the presence of β1–24-corticotrophin and phenylmethylsulphonylfluoride whereas binding was decreased in the presence of EDTA or 8-hydroxyquinoline. 5. These results demonstrate the presence of high-affinity binding sites for AII and of angiotensinases in hepatic membranes.

Solubilization of receptors for pancreatic polypeptide from rat liver membranes

1995 ◽  
Vol 268 (2) ◽  
pp. G215-G223
Author(s):  
T. D. Nguyen ◽  
M. S. Wolfe ◽  
G. G. Heintz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Pancreatic Polypeptide ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Receptor Complexes ◽  
High Affinity

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

scholarly journals Effects of dl-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites

1985 ◽  
Vol 230 (1) ◽  
pp. 169-179 ◽  
Author(s):  
M R Edwards ◽  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Tissue Differences ◽  
The Relationship

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

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.

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