Somatostatin Biosynthesis Occurs in Pancreatic Islets*

Abstract

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.

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 .…”

“…Islet peptides were extracted with 2 M acetic acid as described by Noe et al (13) . Extracts were filtered on columns (1 .6 cm x 90 cm) of Bio-Gel P-10 (100-200 mesh) in 2 M acetic acid at ambient temperature and 1.5-ml fractions were collected .…”

“…Glucagon (fractions 8-11) and desamidoglucagons (fractions [21][22][23][24][25][26] were identified on the basis of tryptophan content and glucagon immunoreactivity . The 2 M acetic acid extraction and gel filtration procedures employed to recover somatostatin (10,13,17) promote selective deamidation of glucagon, which increases its electrophoretic mobility . This accounts for the difference in the pattern shown in Fig .…”

“…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 . Somatostatin is highly basic and migrates to the cathode under the conditions employed (10,13,17) .…”