Characterization of 125I-Glucagon Binding in a Solubilized Preparation of Cat Myocardial Adenylate Cyclase: FURTHER EVIDENCE FOR A DISSOCIABLE RECEPTOR SITE

Abstract

Abstract A solubilized preparation of cat myocardium, which contains adenylate cyclase, has been shown to specifically bind biologically active 125I-glucagon. 125I-glucagon binding was observed over the concentration range 1 x 10-7 to 1 x 10-5 m. Unlabeled glucagon displaced 125I-glucagon over a similar concentration range. The binding specificity of this preparation was also shown by the fact that parathyroid hormone did not bind to cardiac receptor sites in this preparation nor did it displace 125I-glucagon from its binding site. The binding of 125I-glucagon at 37 or 25° is linear for approximately 30 min until maximum binding is reached. In contrast, the activation of adenylate cyclase is maximal within 5 min, indicating the presence of additional glucagon binding sites over and above those required for activation of the enzyme. The binding material was stable at 4° for 4 days and indefinitely when stored in liquid nitrogen. Boiling the binding material for 15 min or incubating it with 1 n HCl for 15 min destroyed most of its glucagon-binding ability. Optimal binding was observed over a broad pH range from 3.6 to 8.5, with a decline above pH 9.0. Preincubation of the binding material with trypsin decreased binding about twothirds. Phospholipases A and C, DNase, RNase, neuraminidase, urea (1 m), GTP, and ATP were without effect on the binding. Solubilized myocardial adenylate cyclase has been shown to have a molecular weight of about 100,000 to 200,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the crude solubilized preparation incubated with 125I-glucagon suggested a molecular weight for the binding fraction of approximately 26,000. Chromatography of the 125I-glucagon-receptor complex on either Sephadex G-100 or Bio-Gel P-30 produced a dissociation of the receptor from catalytic adenylate cyclase activity, which apparently represents a larger molecular weight component (g 100,000). The 125I-glucagon-receptor complex eluted from the gels at an elution volume consistent with the salt peak. However, this eluate was shown by hydrodynamic flow electrophoresis to be neither free glucagon or iodine. Moreover, when it was subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gels, the complex had a molecular weight of about 26,000, identical with what was obtained with the crude material. It appeared, therefore, that the complex of glucagon to its receptor had a strong positive charge which resulted in adsorption of the complex to the gel. Following neutralization of the charge-gel interaction by Sephadex and Bio-Gel chromatography of the 125I-glucagon-receptor complex in 0.01 or 0.025 n NaOH, the binding fraction eluted in a more appropriate location for its apparent size. The property of adsorption to Sephadex G-100 and Bio-Gel P-30 may prove useful in purification of the glucagon receptor site.