title: Evidence of sequential metabolic cleavage of proglucagon to glucagon in glucagon biosynthesis.
abstract: Following a 30 min preincubation in medium containing no isotopes, anglerfish islet tissue was incubated in the presence of [3H]tryptophan and [14C]isoleucine for 20 min. A portion of the tissue was removed for immediate extraction. The remainder was washed thoroughly with unlabeled medium and post-incubated in medium containing an excess of unlabeled tryptophan and isoleucine for varying periods of time. The distribution of radioactive proteins in alcoholic tissue extracts was analyzed by gel filtration and polyacrylamide gel electrophoresis. The distribution of immunoreactive glucagon was determined by radioimmunoassay. Following the 20 min pulse incubation, only proinsulin was labeled with [14C]isoleucine. Two glucagon immunoreactive molecules, one larger than proinsulin (mol wt near 11,400) and the other slightly smaller than proinsulin (mol wt near 9,000), were the primary proteins labeled with [3H]tryptophan following the 20 min. pulse. During chase incubations of increasing duration, 3H-radioactivity appeared in a glucagon immunoreactive molecule with the approximate molecular size of glucagon and increased with chase time while radioactivity in the 11,400 mol wt tryptophan-labeled molecule decreased. With increasing chase time, the 3H-radioactivity attributable to the 9,000 mol wt tryptophan-labeled molecule initially increased and subsequently decreased which is consistent with the pattern that would be expected for a conversion intermediate. The presence of glucagon immunoreactivity in [3H]tryptophan-labeled molecules having molecular weights near that of proinsulin was established by radioimmunoassay of alternate gel slices following electrophoresis of labeled proteins recovered from the proinsulin containing portions of gel filtration eluates. That [14C]isoleucine became incorporated into insulin and [3H]tryptophan became incorporated into glucagon was established by determination of the distribution of radioactivity in polyacrylamide gels following electrophoresis of labeled proteins recovered from the insulin and glucagon containing portions of gel filtration eluates. These results provide preliminary evidence for sequential metabolic cleavage of proglucagon in glucagon biosynthesis.
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism; D005934:Glucagon / Q000096:biosynthesis* / Q000302:isolation & purification; D007328:Insulin / Q000096:biosynthesis; D007515:Islets of Langerhans / Q000378:metabolism*; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D011384:Proinsulin / Q000378:metabolism; D011863:Radioimmunoassay; D014364:Tryptophan / Q000378:metabolism
chemical_list: D007328:Insulin; D007532:Isoleucine; D014364:Tryptophan; D005934:Glucagon; D011384:Proinsulin