Post-translational processing of anglerfish islet somatostatin precursors.

Abstract

Two distinct somatostatin precursors are synthesized in anglerfish (AF) islets. In addition to a precursor which has somatostatin 14 (SS-14) as a C-terminal cleavage product, a precursor which contains at its C-terminus [Tyr7, Gly10] SS-14 as a potential cleavage product is also synthesized. However, even though an Arg-Lys pair is located immediately N-terminal to Ala1 of the C-terminal tetradecapeptide, [Tyr7, Gly10]SS-14 was not found in significant amounts in extracts of AF islets. Instead, a 28 residue peptide having [Tyr7, Gly10]SS-14 (AF SS-28) at its C-terminus was found to be a primary cleavage product of this form of pro-SS. A question which arises from these observations is whether the differential cleavage of pro-SS-14 (PSS-I) and pro-SS-28 (PSS-II) is the result of differences in primary and/or secondary structure of the two precursors which in turn modulate the activity of the same converting enzyme, or whether separate cleavage enzymes exist for each precursor. Experiments were designed to address this question. Microsomes (M) and secretory granules (SG) were isolated from AF pancreatic islets. Fraction purity was monitored by RIA for islet hormones, and by assays for plasma membrane and lysosomal enzymes. The ability of lysed M and SG preparations to mediate conversion of radiolabeled islet prohormones to products was monitored by gel filtration and HPLC analysis of the products. The pH optimum for converting activity in M and SG was found to be near 5.0. Incubations in the presence of selective proteinase inhibitors and prohormones containing Arg and Lys analogs demonstrated that a cysteine proteinase(s) which cleaves at basic amino acid residues is involved in granule-mediated conversion. A significant proportion of the converting activity in granules was found to co-precipitate with SG membranes. Washing these membranes with 1M KC1 resulted in dissociation of most of the converting activity from the membranes suggesting that the proteinase(s) involved is membrane-associated. The processing activities for proinsulin and pro-SS-28 which were observed in SG were also found to be active, and membrane-associated, in M. However, converting activity for pro-SS-14 was found only in SG. Much of the PSS-I to SS-14 processing activity was membrane-associated in SG. By contrast, pro-SS-28 converting activity in SG was entirely soluble. These results suggest that two or more separate enzymes are involved in processing pro-SS-14 and pro-SS-28 and that these enzymes have differential activity in M and SG.(ABSTRACT TRUNCATED AT 400 WORDS)