Molecular and immunological properties of insulin-like and proenkephalin-like material from nervous tissue: implications for neuropeptide biosynthesis

Abstract

The studies reported in this thesis concerned the biosynthesis and processing of two neuropeptides, insulin and proenkephalin. 1) Two main forms of immunoreactive insulin were characterized from cultures of mouse foetal brain cells. The major component was converted by trypsin to the minor form, which resembled authentic pancreatic insulin in chromatographic behaviour. Both components were found to have similar immunological and chemical properties to those of insulin and proinsulin. 2) The insulin-like species present in foetal brain cell cultures was shown to possess insulin activity in a biological assay based on the incorporation of [3H]-glucose into lipids of isolated rat epididymal fat cells. This assay permitted the detection of other insulin-like activities unrelated to immunoreactive insulin. One of these components was present in very high concentrations in culture medium and was identified as immunoreactive IGF-1. 3) Antisera to a synthetic peptide which corresponded to residues 95-117 of bovine proenkephalin were shown to recognize all major enkephalin-containing intermediates in the bovine adrenal medulla. These antisera enabled characterization of the molecular forms of proenkephalin in this tissue by gel filtration followed by radioimmunoassay and SDS-gel electrophoresis and subsequent immunoblotting. A greater diversity of molecular forms was detected than previously reported. Each major immunoreactive form was resolved into at least two closely related forms by SDS-polyacrylamide gel electrophoresis. 4) A high molecular weight proenkephalin intermediate (27-kDa) was found to be associated with the bovine adrenal medullary chromaffin granule membrane and it was also present in soluble form. High concentrations of chaotropic agents were required to extract the membrane-associated proenkephalin. 5) Enzymatic processing of high molecular weight proenkephalin intermediates was studied by SDS-polyacrylamide gel electrophoresis and immunoblotting with antisera that recognized proenkephalin. Preliminary results indicated specific processing of the soluble 27-kDa proenkephalin intermediate by an enzyme with a thiol group present in the active site. Some evidence was obtained for the regulation of processing enzyme(s) by soluble factors in chromaffin granules. There appeared to be differences between the processing of the membrane-associated 27-kDa material and that of the soluble form 6) The molecular distribution of proenkephalin (95-117) immunoreactivity was determined in regions of rat brain. Region-specific processing was observed with marked variations in the gel filtration profiles from different brain regions. Rat neuronal proenkephalin appeared to be highly processed with low concentrations of intermediates and high concentrations of free enkephalins. The distribution of immunoreactivity in rat adrenal glands and brain was different and suggested differences in processing by these tissues. 7) Multiple forms of proenkephalin (95-117) immunoreactive material were characterized from mouse foetal cell cultures, a neuroblastoma x glioma cell line and perinatal hypothalamic cell cultures by gel filtration and h.p.1.c. Two forms of immunoreactive met-enkephalin were also extracted and characterized from mouse foetal neuronal cell cultures; these behaved differently from met-enkephalin on h.p.1.c. Enzymatic digestion with trypsin and carboxypeptidase B converted both species to a third met-enkephalin immunoreactive form. All three species appeared to represent N-terminally extended forms of met-enkephalin which were not found in intact foetal brain.