Date of Award
Doctor of Philosophy (PhD)
R. M. Epand
A heterobifunctional crosslinking reagent 2-nitro-4-azido-phenylsulfenyl chloride (NASPCl) was synthesized and characterized. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan containing polypeptides and proteins lacking sulfhydryl groups. The reaction of NAPSCI with glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine gave one major product which could be effectively photolysed at wavelengths above 300 nm. Glucagon-NAPS could be radiolabeled by the lactoperoxidase catalyzed iodination of the peptide. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS upon photolysis was demonstrated. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes with a slightly higher potency than the native hormone at equimolar concentrations. Glucagon-NAPS was, therefore, considered as an effective photoaffinity probe for labeling the glucagon receptor sites in plasma membranes with a target cells.
Irradiation of hepatocyte plasma membranes in the presence of glucagon-NAPS resulted in an activated state of adenylate cyclase. The enzyme displayed a lower response to further stimulation by native glucagon, glucagon-NAPS or NaF than the response displayed by similarly treated membranes in the dark, suggestive of covalent labeling of the derivative to glucagon functional sites on the membrane.
Competitive binding studies at steady state using radio-labeled ¹²⁵I-glucagon and ¹²⁵I-glucagon-NAPS demonstrated binding of glucagon-NAPS to the same receptor sites on hepatocyte plasma membranes as native glucagon. Scatchard plot analysis of the binding isotherm curves indicated two orders of specific receptor sites: a) a high affinity-low capacity site, (glucagon, Kd = 3.52 (± 0.72) x 10ˉ¹⁰M, Bmax = 0.34 ± 0.15 pmole/mg membrane protein; glucagon-NAPS, Kd = 1.31 (± 0.10) x 10ˉ¹⁰M, Bmax = 0.67 ± 0.10 pmole/mg membrane protein) and b) a low affinity-high capacity site, (glucagon, Kd = 4.17(± 1.05) x 10ˉ⁹ M, Bmax = 3.08 ± 0.63 pmole/mg membrane protein; glucagon-NAPS, Kd = 7.28(± 1.11) x 10ˉ¹⁰M, Bmax = 2.19 (± 0.95) pmole/mg membrane protein). The binding characteristics of glucagon and glucagon-NAPS were similarly affected by guanosine 5'-triphosphate (GTP). Saturation of the binding sites at concentrations of glucagon and glucagon-NAPS producing maximal stimulation of adenylate cyclase suggested the requirement of interactions with both sites for a functional adenylate cyclase system. Hill plot analysis indicated noncooperative interactions of the peptides with the high affinity-low capacity sites and cooperative interactions with the low affinity-high capacity sites.
Covalent crosslinking of bound ¹²⁵I-glucagon-NAPS to hepatocyte plasma membranes upon irradiation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified a number of radiolabeled membrane components of which a 67,000-70,000 daltons component was significantly labeled above background and was displaced by more than 90% in the presence of excess unlabeled glucagon or glucagon-NAPS. This membrane component was identified as the glucagon specific binding site. The apparent molecular weight of detergent solubilized glucagonreceptor complex in a GTP sensitive state was estimated by gel fractionation as 200,000-250,000. SDS-gel electrophoresis of detergent solubilized, ¹²⁵I-glucagon-NAPS-receptor complex, covalently crosslinked by irradiation, identified the 67,000-70,000 as well as a 50,000 and 27,500 daltons components, also observed in unsolubilized membranes. Since the latter components were not significantly labeled above background or displaced in membrane samples irradiated in the presence of unlabeled peptide, they may not be called specific receptor sites. The inhibition of covalent crosslinking of all three components in trypsinized membranes demonstrated their protein nature. Anomalous electrophoretic mobilities with different acrylamide concentrations were suggestive of the receptor peptide being a glycoprotein. Similar behaviour under reducing and nonreducing conditions was suggestive of the presence of intramolecular disulfide bonds.
In these studies we have demonstrated that the glucagon-NAPS derivative has the advantage for labeling the glucagon receptor in hepatocyte plasma membranes because it is biologically active and the binding sites for the derivative can be linked to a physiological response. The receptor site identified should represent the true glucagon receptor or a receptor subunit since upon irradiation it was covalently crosslinked by the ligand, with the receptor still being in a biologically active state.
Demoliou, Catherine Demetriou, "The Preparation and Characterization of a Photoreactive Glucagon Analogue and its Interaction with Rat Liver Plasma Membranes" (1980). Open Access Dissertations and Theses. Paper 3313.