Purification and Biosynthesis of Canine Gastric Glucagon

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(1) Characterization and purification of canine gastric glucagon. Among the gastrointestinal tissues of the dog, upper stomach contains the highest concentration of gastrointestinal glucagon (IRG). In order to purify and characterize it, IRG was routinely extracted from mucosal scrapings of the fundus of the stomach from 5 dogs, using an acid-alcohol solution containg 0.01 M benzamidine. Ether-ethanol precipitates of these acid alcohol extracts were subsequently purified by gel-filtration and ion-exchange chromatography. The protein content in the coulumn effluent was monitered at either 280nm or 254nm. IRG was measured using two antisera; 30-K, a serum highly specific for pancreatic glucagon (PG), and K-4023 which strongly crossreacts with gut glucagon. IRG was eluted as one major peak corresponding to PG by gel-filtration on Bio-gel P 30 in 3M acetic acid and then on the same gel in 0.05 M NH4HCO3. Immunoassay dilution curve of the major peak was parallel to the dilution curve of PG standard. The IRG peak obtained after the second gel-filtration was resolved by polyacrylamide gel disk electrophoresis (PGDE) in urea at pH 8.7 into three immunoreactive components. The main component was identical to PG in its mobility. The faster minor component was very likely desamido-glucagon. A slower component, barely entering the gel, reacted more strongly with K-4023 and therefore presumably contains hydrolized fragments of glucagon. Thus the IRG peak obtained after gel-filtration was not immunologically homogeneous. Therefore the IRG peak from the alcaline Bio-gel coulumn was rechromatographed on an ion-exchange column DEAE-Sephadex A-25. Two or three immunoreactive peaks were obtained. The peak, which had the highest specific immunoreactivity was eluted similarly to the PG. This major IRG peak was then subjected to PGDE. Only one immunoreactive component was identfied and it reacted equally well with the specific antiserum 30-k and the nonspecific K-4023. This indicates that this IRG component from the ion-exchange column was immunologically pure. When the gel was stained with coomassie-blue one stainable band was seen which has an electrophoretic mobility similar to standard PG. This peak was also tested for receptor binding activity using liver plasma membrane and it was found that the displacement of I125-glucagon by serial dilution of this material was similar to that obtained with standard PG. The IRG peak from the ion-exchange column DEAE-Sephadex A-25 was rechromatographed on an ion-exchange coulmn CM-Bio-gel A. Only one immunoreactive peak was obtained. This IRG peak is tested its biological activity. (2) Biosynthesis of glucagon in canine gastric mucosa. 2g of gastric mucosa were incubated in 3 ml Krebs-Ringer bicarbonate buffer which contained trasylol and antibiotics, gassed with 95% O2-5% CO2 at 37°C for 5 hrs in the presence of 3H-tryptophan. Gastric mucosa was rinsed with Krebs-Ringer bicarbonate buffer, then homogenized in 10% trichlor acetic acid added 10mg of porcine pancreatic glucagon (Lilly). IRG was extracted and purified by the same method discribed above. One of the peaks of 3H-tryptophan was observed in the PG region. PGDE of this peak showed that one major peak of 3H-tryptophan was comigrated with PG.
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Purification and Biosynthesis of Canine Gastric Glucagon

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