Two molecular forms of insulin from barfin flounder, Verasper moseri, are derived from a single gene

We have purified and characterized two molecular forms of insulin from the Brockmann bodies of barfin flounder, Verasper moseri: a normal type of insu lin (insulin-I), which consisted of 21 amino acid residues for the A-chain and 30 residues for the B-chain, and a novel type of insulin (insulin-II), which had an extension of two amino acid residues at the N-terminus of the B-chain. The additional two residues at the N-terminus of B-chain of insuli n-II were Pyr-Ala which had not yet been reported in vertebrate insulins. E xcept for these two residues, the amino acid sequence of insulin-it was com pletely identical with that of insulin-I. Each Brockmann body extract from five individuals contained both insulins, indicating that insulin-I and -II were the products of non-allelic expression. By polymerase chain reaction, only one nucleotide sequence of preproinsulin gene encoding insulin-I and -II was obtained, and the amino acid sequence of A- and B-chains deduced fr om the nucleotide sequence was identical with that of insulin-I and also in sulin-II established by Edman degradation. Furthermore, genomic Southern bl ot analysis using a part of nucleotide sequence of the preproinsulin gene a s a probe showed a single positive band in ail cases of genomic DNA digeste d with each of three restriction endonucleases. These results indicate that insulin-I and -II of barfin flounder arise from a single preproinsulin by proteolytic cleavage at different sites of the signal peptide region.