Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species.

Abstract

Fetal bovine pancreas was extracted for glucagon using (A) ethanol-Hcl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol-HCl and (C) urea-acetic acid. Fractionation of the acetic acid soluble proteins vi Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" (approximately equal to 9 K delta), and true glucagon (3.5 K delta) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol-HCl-TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to approximately 40 K delta and approximately 12 K delta. HMW-IRGs did not bind to concanavalin A (Con A)-agarose. SDS-polyacrylamide gel electrophoresis of the Con A-agarose filtered IRG again showed a major immunoreactive peak of approximately 40 K delta. Dose-response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS-polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight greater than 20 K delta) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).