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.

The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.

No information is at present available on the mode of SRIF biosynthesis. Since anglerfish pancreatic islet tissue is comprised of approximately 30% D cells, we have examined this tissue for SRIF synthesis . The following known differences in amino acid composition of islet peptides were used advantageously in this study: anglerfish proinsulin: Trp-0, Ile-2, Cys-6; anglerfish glucagon: Trp-1, Ile-0, Cys-0; mammalian SRIF: Trp-1, Ile-0, Cys-2. After incubating islet tissue with [3H]tryptophan and [14C]isoleucine or [35S]cystine for various time periods, proteins were extracted in 2 M acetic acid and desalted by Bio-Gel P-2 gel filtration. P-2 void volume proteins were then subjected to P-10 gel filtration and isolated by polyacrylamide gel electrophoresis (PAGE) at alkaline pH. The predominant amount of the immumoreactive SRIF in the extracts appeared in a peak eluting just before the salt volume on P-10 filtration and migrated slowly toward the cathode during PAGE. The behavior of synthetic SRIF was identical. The anglerfish SRIF immunoreactive peptide could be labeled with Trp and Cys but not Ile during incubations longer than 1 h. The Trp- and Cys-labeled peptide could be bound on columns to which the immunoglobulin fraction of antisera to SRIF had been complexed. Cycloheximide inhibited isotope incorporation into all islet proteins. These results indicate that islet SRIF is synthesized in situ. Moreover, the immunological activity, size, and charge characteristics of anglerfish islet SRIF appear to be similar to those of mammalian hypothalamic SRIF. When islets were subjected to short pulse incubations with labeled Trp and Cys, only peptides eluting in the 7,000-13,000 dalton portion of the filtration eluate became labeled. No appreciable isotope incorporation into SRIF was observed. However, when pulse incubations were followed by incubation in the presence of cycloheximide or excess unlabeled amino acids in isotope-free medium (chase), the incorporation of Trp and Cys into SRIF increased with the length of chase, suggesting the participation of a larger precursor in SRIF synthesis.