Characterization of the Conversion of a Somatostatin Precursor to Somatostatin by Islet Secretory Granules

Abstract

The existence of a biosynthetic precursor for somatostatin has been previously demonstrated in angler-fish pancreatic islet tissue. In the present study, the subcellular conversion of this precursor to somatostatin was examined. When subcellular fractions were assayed for immunoreactive somatostatin, 86% of the immunoreactivity was found in the microsome and secretory granule fractions. Based on this finding and results from previous studies on proglucagon and proinsulin conversion, only these two fractions were examined for converting activity. After incubating islet tissue with [³H]tryptophan and [³⁵S]cystine or [¹⁴C]iso-leucine for varying time periods, the tissue was fractionated and the microsome and granule fractions were subjected to postincubation. Conversion was monitored by the calculation of percent accumulation of somatostatin in eluates after Bio-Gel P-10 filtration of fraction extracts. Both secretory granules and microsomes converted endogenously labeled 8000–15,000-dalton precursor peptides to somatostatin. Disruption of the granules by repeated freeze-thawing or osmotic shock did not affect converting activity. Conversion of exogenously labeled precursors was also observed in both fractions. When granules were subjected to repeated freeze-thawing and high-speed centrifugation to separate membranes from sol, the membranes were found to have 29% more converting activity than the resulting supernates. Both membranous and soluble converting activities were significantly inhibited in the presence of leupeptin but not phenylmethyl sulfonyl fluoride. Significant inhibition of conversion was observed when lysed granules were postincubated in the presence of antipain, leupeptin, and p-chloromercuribenzoate but not chloroquine, di-isopropyl fluorophosphate, EDTA, or N-p-tosyl-L-lysine-chloromethyl ketone HCI. These results indicate that the prosomatostatin converting enzyme is a unique membrane-associated thiol proteinase with a possible specificity for arginine residues. The findings also indicate that the intracellular transport and processing of prosomatostatin is similar to that of other peptide hormone precursors.