Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species
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 via Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" [Formula: see text], and true glucagon (3.5 K Δ) 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 [Formula: see text] and [Formula: see text]. 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 [Formula: see text]. 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 > 20 K Δ) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).