Higher molecular weight forms of immunoreactive somatostatin in mouse hypothalamic extracts: Evidence of processing in vitro

AUTOR(ES)

Lauber, Marc

RESUMO

Extracts of mouse hypothalamus made in acid/urea containing protease inhibitors were analyzed for somatostatin immunoreactivity after molecular sieve filtration on Sephadex G-50. Higher molecular weight (higher-Mr) somatostatin-like forms with apparent molecular weights of 15,000, 10,000, and 6000 could be identified, besides the molecular weight 1600 somatostatin. Immunological identities with somatostatin were unambiguously demonstrated by the analysis of the displacement curves in the radioimmunoassay. The Mr 15,000, 6000, and 1600 species were purified by affinity chromatography on an anti-somatostatin immune serum covalent conjugate with Sepharose used as immunoadsorbant. After disulfide reduction by dithiothreitol, the size of the Mr 15,000 and 6000 somatostatin-like species was assessed either by molecular sieve filtration or by polyacrylamide gel electrophoresis. The results indicated that the higher-Mr somatostatin-like species isolated from the hypothalamus did not result from hormone polymerization by means of disulfide interchange. The processing in vitro of the 15,000 higher-Mr form of somatostatin was achieved by proteolytic enzymes coeluted with this species during the fractionation of hypothalamic extracts. Under neutral pH conditions the intermediary higher-Mr forms were generated together with the Mr 1600 somatostatin-like species. This processing activity could be either strongly inhibited at acidic pH or in acid/urea medium or else eliminated by selective immunoadsorption of the 15,000 higher-Mr form. Neither trypsin nor the γ subunit of 7S nerve growth factor was able to produce this processing, suggesting that enzymes with other kinds of specificity may be involved. It is concluded that somatostatin biosynthesis in the mouse hypothalamus may occur via a high-Mr precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.

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