Abstract
Abstract The glucagon immunoreactivity present in canine pancreatic extracts can be separated by gel filtration into two fractions; one fraction, comprising over 90% of the immunoreactivity, is similar in molecular size to the glucagon-131I marker, while the other appears to be at least twice as large. Incubation of this large glucagon immunoreactivity (LGI) in solutions such as 1 m acetic acid, 8 m urea, 0.1 m mercaptoethanol, or 6 m guanidine hydrochloride, which favor the dissociation of noncovalent protein complexes, fails to change its molecular size as determined by gel filtration, suggesting that LGI is not glucagon noncovalently bound either to itself or to another protein. However, tryptic hydrolysis of LGI results in the disappearance of most of the immunoreactivity and in a reduction in the molecular size of the remaining immunoreactivity to approximately that of glucagon-131I. Neither LGI nor its tryptic product, both of which resemble glucagon immunologically, has glycogenolytic activity in the perfused rat liver. It is possible that LGI includes the immunoreactive sequence of glucagon or of an immunologically similar peptide.
Highlights
Chromatography of Canine Pancreatic Extracts-The elution pattern of extractable glucagon immunoreactivity of the canine pancreas from Bio-Gel P-10 columns is shown in Fig. lA, which is representative of eight such experiments
To determine whether spontaneous dissociation of large glucagon immunoreactivity (LGI) occurs under these conditions, a concentrated pool of eluates containing 6 mg of protein and 150 to 300 mpg eq of LGI in 0.6 ml was rechromatographed on a Bio-Gel P-10 column in NHbHC03 solution at pH 8.8, as described under ‘Methods and Materials.”
These findings suggest that large glucagon immunoreactivity is devoid of glycogenolytic activity in vitro and in vivo
Summary
Eluates were assayed for protein by means of absorbance at 280 rnp in a DU spectrophotometer or by the method of Lowry et al (7), or both. Glucagon immunoreactivity of eluates was determined by the previously described radioimmunoassay (8), in which antisera highly specific for pancreatic glucagon were used. Acetic acid, or guanidine hydrochloride, were either dialyzed at 4” for 48 hours against 0.025 M NHJHCO~ at pH 8.8 or diluted to concentrations which could not influence the assay. Beled markers were determined by assay of eluates for radioactivity. Glucose concentration of samples of liver perfusate and of canine plasma was determined by the method of Hoffman (9) with the use of the Technicon autoanalyzer
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