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.

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.

Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
Author(s):  
Christie Cefaratti ◽  
Andrea Romani ◽  
Antonio Scarpa
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Channel Blockers ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Distribution of opioid receptors in canine small intestine: implications for function

1989 ◽  
Vol 256 (6) ◽  
pp. G966-G974 ◽  
Author(s):  
H. D. Allescher ◽  
S. Ahmad ◽  
P. Kostka ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Sites ◽  
Myenteric Plexus ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Opiate Receptors ◽  
Circular Muscle ◽  
Maximum Binding Capacity ◽  
Opiate Binding ◽  
Subtype Selective

Distribution of the binding sites for [3H]diprenorphine, a non-selective opiate ligand, was studied in membrane fractions from longitudinal muscle/myenteric plexus and circular muscle containing deep muscular plexus. [3H]saxitoxin was used as a marker for neuronal plasma membranes and 5'-nucleotidase as a marker for smooth muscle plasma membranes. Saxitoxin binding correlated strongly with diprenorphine binding, but 5'-nucleotidase correlated poorly with diprenorphine or saxitoxin binding in these fractions. Opiate binding sites in membranes of myenteric and deep muscular plexus were of high affinity (Kd = 0.12 and 0.18 nM, respectively) with maximum binding capacity of 400 and 500 fmol/mg protein, respectively. Competition experiments using subtype-selective opiate ligands indicated that all three subtypes of opiate receptors were present in the same ratio of 40-45% mu-subtypes, 40-45% delta-subtypes, and 10-15% kappa-subtypes on both plexuses. Opiate receptors of canine small intestine, therefore, are located primarily or exclusively on nerves with similar distributions in nerve membranes containing only axonal varicosities (deep muscular plexus) as in those containing neurons, dendrites, and varicosities (myenteric plexus).

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes

1997 ◽  
Vol 152 (3) ◽  
pp. 407-412 ◽  
Author(s):  
M Montiel ◽  
M C Caro ◽  
E Jiménez
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Ang Ii ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5. Journal of Endocrinology (1997) 152, 407–412

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.

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