The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. 3. Binding of glucagon: method of assay and specificity.

The initial reaction between glucagon and the glucagonsensitive adenyl cyclase system in rat liver plasma membranes is probed with the use of *2SI-glucagon. An assay system is described for the binding of labeled glucagon to membranes. Iodination of glucagon with Ip yields mono-iodo glucagon having the same biological activity as native glucagon. 1251Glucagon of high specific activity used for studying binding cochromatographs with native glucagon and behaves as native glucagon according to the following observations. (a) Biological activity and binding of labeled glucagon are reduced in parallel and to the same extent by a glucagon inactivating process in the liver membrane preparation; at high membrane concentrations, all of the medium glucagon is inactivated rapidly; (b) binding of labeled glucagon is reduced specifically by native glucagon in proportion to the relative concentration of labeled and unlabeled glucagon added; and (c) binding of glucagon is not altered in the presence of biologically inactive peptide fragments of glucagon (Fragments 1-21 and 22-29), secretin, adrenocorticotropin, and insulin. Products of glucagon degradation formed during iodination are also biologically inactive and do not bind to liver membranes. Liver plasma membranes bind 20 times more labeled glucagon than do fat cell ghosts which contain a glucagonsensitive adenyl cyclase system that has apparent atEnity for glucagon 25-fold less than the liver membrane system (4 x lop9 M glucagon). The apparent affinity of the liver membrane binding sites for glucagon is approximately the same as that of the adenyl cyclase system for glucagon. The binding sites for glucagon are finite in number (estimated 2.6 pmoles per mg of membrane protein) and are saturated with hormone in the range of 4 to 8 X lo-* M glucagon, the same range found for maximal activation of adenyl cyclase by the hormone. Treatment of liver membranes with phospholipase A, digitonin, and urea cause parallel losses in binding of glucagon and activation of adenyl cyclase by the hormone. Combined with the similar range of concentrations over which glucagon binds and activates adenyl cyclase, the correlations