The present studies were designed to obtain further information concerning the chemical interrelationships of the fractions of glucagon-like immunoreactivity (GLI) present in gastrointestinal tissues, their biologic activity, and their immunochemical and electrophoretic properties. Chromatography of canine jejunal extracts on a Bio-Gel P-10 column separated GLI into two fractions; one, designated Peak II, eluted with the glucagon-I-131 marker (molecular weight 3,500), while the other, designated Peak I, appeared before the insulin-I-131 marker (molecular weight >6,000). Neither rechromatography, nor incubation in 8 M urea, acetic acid, or mercaptoethanol changed the elution volume of Peak I; however, trypsin caused a major loss of immunoreactivity and a shift of residual immunoreactivity to the postglucagon-I-131 zone.

Peak I was devoid of glycogenolytic activity in the perfusedrat liver, as was its tryptic product, while Peak II was consistently as active as pancreatic glucagon. Immunoprecipitated Peak II was also active, proving that the immunoreactive fraction was responsible for its biologic action.

Unlike pancreatic glucagon, Peak I, Peak II, and the tryptic product of Peak I diluted disproportionately with the less specific antiserum; however, Peak II could not be differentiated from pancreatic glucagon by means of polyacrylamide gel disc electrophoresis.

It is concluded that (1) Peak I does not consist of Peak II bound noncovalently either to itself or to another protein; (2) Peak I and its tryptic product are devoid of glycogenolytic activity while Peak II has the approximate activity of pancreatic glucagon; (3) the glycogenolytic and immunoreactive components in the eluate containing Peak II are probably on the same molecule; (4) both Peak I, its tryptic product, and Peak II are immunologically different from pancreatic glucagon.

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