Iodoglucagon. Preparation and Characterization

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Abstract
Iodinated derivatives of glucagon containing an average of 1 to 5 g-atoms of 127I per mol have been prepared by reacting the hormone with increasing amounts of iodine monochloride. Their iodoamino acid composition has been determined by ion-exchange chromatography and electrophoresis, following hydrolysis by pronase. Iodination of the two tyrosyl residues occurs first and is nearly complete after addition of a 4-fold molar excess of ICI. Iodination of the single histidyl residue is a later event and does not exceed an average of one atom per residue. Hydrolysis of iodoglucagon by trypsin and subsequent separation of the iodotyrosyl peptides shows that iodine is equally distributed between tyrosyl residues 10 and 13. Crude iodoglucagon containing an average of 1 g-atom of iodine per mol has been resolved into several components of differing iodine content and iodoamino acid composition by chromatography on DEAE-cellulose. Monoiodoglucagon isolated by this procedure shows a single band when analyzed by polyacrylamide gel electrophoresis. Iodoglucagons containing an average of 1 to 4 g-atoms of iodine per mol are more potent than native glucagon in their ability to stimulate adenylate cyclase activity and to bind to glucagon receptors of liver cell membranes of the rat. The maximal increase in biological potency occurring upon iodination is about 5-fold with respect to adenylate cyclase activity, and 2-fold with respect to binding to receptors; tetra and triiodinated derivatives show, respectively, the highest potency. Similar effects occur whether inactivation by liver membranes is inhibited or not, indicating an enhancement in the intrinsic affinity of iodoglucagon for the receptors. Iodination beyond 4 g-atoms per mol slightly decreases the affinity of the hormone for adenylate cyclase and for the receptors. Iodination causes a 2–20-fold decrease in the ability of liver plasma membranes and of blood plasma to inactivate glucagon in vitro; these effects correlate with the degree of iodination. With liver microsomal membranes, a decrease in glucagon inactivation occurs only at iodine contents exceeding 4 g-atoms per mol, and lower degrees of iodination result in opposite effects. Monoiodination causes a 4–6-fold increase in the plasma concentration of glucagon within the first 18 min following a single intravenous injection of the hormone to rats. More extensive iodination results, in addition, in a marked decrease in the rate of disappearance of glucagon from the blood. The immunological reactivity of glucagon is little affected by monoiodination, but strongly depressed by higher degrees of iodination. Iodination of glucagon with chloramine-T results in derivatives that are less potent than those prepared with ICI in their ability to activate adenylate cyclase and to bind to the receptors. With this procedure little or no increase in the affinity of glucagon for the receptors occurs. Chloramine-T alone decreases the affinity of the glucagon-receptor interaction. Highly radioactive, 125I-labeled derivatives of glucagon containing an average of 1 g-atom of iodine per mol show little increase in their ability to activate adenylate cyclase. With increasing degrees of iodination, such derivatives show less affinity for the receptors, and become more resistant to inactivation or to hydrolysis to free iodotyrosines upon exposure to liver membranes.