Evidence for 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.

Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

“…2 C) . The elution patterns obtained at pH 5 .2 were indistinguishable from those obtained from extracts of intact tissue (7,10,13,14), or secretory granules isolated after pulse incubation and protected from lysis by suspension in 0 .25 .M sucrose during 6-to 8-h chase incubations . The data on hormone accumulation, obtained after incubation of lysed secretory granules in buffers at six different pH values, are summarized in Fig.…”

“…However, for the purposes of these experiments, this is actually advantageous . All ofthe anglerfish islet prohormones are slightly larger in molecular size than porcine proinsulin (7,13,14,16,17) . Therefore, 1 frg ofporcine proinsulin should contain more dibasic residues (more molecules) for converting enzyme recognition than 1 hg of the anglerfish islet prohormone mixture .…”

“…Using ["C]isoleucine or reduced ['S]cystine, anglerfish proinsulin and insulin are selectively labeled and clearly defined as separate "C-or "S-labeled peaks in Bio-Gel P-10 filtration eluates; glucagon lacks both amino acids andsomatostatin lacks isoleucine (l3, 14). Percent accumulation of insulin was obtained by summation of the dpm in the proinsulin and insulin peaks, then use of the formula : % accumulation of insulin = [( 1 "C or 35S dpm insulin)/('"C or "S dpm proinsulin + insulin)] x 100.…”

“…This accounts for the difference in the pattern shown in Fig . 5 A as compared with those obtained when extraction is in acid ethanol and gel filtration is performed using 1 M acetic acid (14,15) . The enhanced mobility is in accordance with the results of Bromer et al (24), who examined the electrophoretic mobility of deamidated glucagons at high pH using 200Io acrylamide gels .…”

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

“…Our results also indicate that the incorporation of radioactive tryptophan into polypeptides of smaller molecular weight increases with the period of incubation, more than 2 h of incubation being necessary to obtain a significant amount of radioactive immunoreactive glucagon. Delayed incorporation of labelled amino-acids into pancreatic precursors and glucagon has also been observed; thus the appearance of label in 9 K polypeptide occurs after 2 h in angler fish islets [26] and after 4 h in the perfused rat pancreas [27]. Appearance of the label in glucagon takes more than 4 h in the perfused rat pancreas [27] and more than 6 days in cultured guinea pig islets [28].…”