Showing papers by "Richard M. Epand published in 1982"
TL;DR: Results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds and show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptors and thus aid in its further characterization.
Abstract: The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.
19 citations
TL;DR: The concentration-dependent stimulation of adenylate cyclase by the photoaffinity reagent 2-[(2-nitro-4-azidophenyl)sulfenyl]Trp25-glucagon (Glucagon-NAPS) and also its binding characteristics were compared with those of the native hormone to suggest noncooperative interactions.
Abstract: The concentration-dependent stimulation of adenylate cyclase by the photoaffinity reagent 2-[(2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon (glucagon-NAPS) and also its binding characteristics were compared with those of the native hormone. The derivative was found to be slightly more potent in stimulating adenylate cyclase than glucagon, in the presence of guanosine 5'-triphosphate (GTP). 125I-Labeled glucagon-NAPS or 125I-labeled glucagon bound rapidly to receptors and was competitively displaced by unlabeled glucagon or glucagon-NAPS. Glucagon-NAPS displaced bound radiolabeled hormone at a lower concentration than did glucagon in the absence of GTP. Scatchard analysis of the binding data obtained from displacement of bound radiolabeled ligand with unlabeled peptide demonstrated a heterogeneous population of saturable glucagon binding sites. Glucagon-NAPS displayed a higher affinity (0.7 nM) for the high-capacity sites (80-90% of total binding sites) than glucagon (4.0 nM) in the absence of GTP. In the presence of the nucleotide, both ligands had approximately the same affinity (0.5-0.6 nM). Hill plot analysis of the binding data suggested noncooperative interactions. Photoaffinity labeling of plasma membranes with glucagon-NAPS resulted in an irreversible activation of adenylate cyclase with a reduced response to further stimulation by glucagon, glucagon-NAPS, and NaF.
14 citations
TL;DR: It is unlikely that the association of apolipoprotein A-I with dimyristoylphosphatidylcholine is thermodynamically stable only in the temperature region of the phase transition but that the relationship exhibits a large kinetic stability, especially at lower temperatures or in the absence of guanidinium chloride.
13 citations
TL;DR: The maximal stability of the glucagon-lipid complex at the phase transition of the lipids indicates that the glucagons-lipids interaction is highly dependent on the structural organization of the lipid.
9 citations
TL;DR: The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their tipolytic activities with the exception of the glycin-amide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenYL cyclase than in lipolysis.
Abstract: Glucagon 1-6 has a maximum lipolytic activity (Lmax) in the rat adipocyte which is 66% of that of glucagon. The N epsilon-guanidyl derivative, modified at Lys12, has about the same Lmax as glucagon 1-6. Modifying the carboxyl groups of glucagon with glycinamide or removing the COOH-terminal residues with cyanogen bromide reduces Lmax to less than 25% of the level of glucagon. The potency of each of these analogs (A50) in microM is as follows: glucagon 6 X 10(-3); glucagon 1-6 2 X 10(-2); N epsilon-guanidyl glucagon 9 X 10(-3); glycinamide glucagon 10(-2); cyanogen bromide peptide of glucagon 2 X 10(-1). The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their lipolytic activities with the exception of the glycinamide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenyl cyclase than in lipolysis. Glucagon 1-6 is much more potent in stimulating adipocyte than liver adenyl cyclase.
6 citations
TL;DR: The hydrophobic myelin protein, lipophilin, has been incorporated into bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol and it was found that a molecule of the protein could prevent an increase in volume at the phase transition temperature of 19 lipid molecules.
5 citations
TL;DR: The trinitrophenyl (TNP) derivative of glucagon has less [ipolytic activity and potency] than the carbamyl derivative, but all the derivatives stimulate this enzyme in the adipocyte.
Abstract: The trinitrophenyl (TNP) derivative of glucagon has less [ipolytic activity and potency than the carbamyl derivative. TheNα,e-acetyl derivative has slightly less activity than the TNP derivative. In contrast to liver, where the TNP derivative fails to stimulate adenyi cyciase, all the derivatives stimulate this enzyme in the adipocyte.
3 citations
TL;DR: The results suggest that the N-terminal histidine of glucagon is involved in both binding to plasma membranes and transduction of the signal to adenylate cyclase.
2 citations