Lauber M, Camier M, Cohen P
Proc Natl Acad Sci U S A. 1979 Nov;76(11):6004-8. doi: 10.1073/pnas.76.11.6004.
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-M(r)) 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 M(r) 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 M(r) 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-M(r) 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-M(r) 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-M(r) forms were generated together with the M(r) 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-M(r) form. Neither trypsin nor the gamma 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-M(r) precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.
将含有蛋白酶抑制剂的酸/尿素制成的小鼠下丘脑提取物，在Sephadex G - 50上进行分子筛过滤后，分析其生长抑素免疫反应性。除了分子量为1600的生长抑素外，还可鉴定出表观分子量为15000、10000和6000的较高分子量（较高M(r)）的生长抑素样形式。通过放射免疫分析中置换曲线的分析，明确证明了与生长抑素的免疫同一性。M(r)为15000、6000和1600的物质通过在与琼脂糖共价偶联的抗生长抑素免疫血清上进行亲和层析纯化，该琼脂糖用作免疫吸附剂。用二硫苏糖醇进行二硫键还原后，通过分子筛过滤或聚丙烯酰胺凝胶电泳评估M(r)为15000和6000的生长抑素样物质的大小。结果表明，从小鼠下丘脑分离出的较高M(r)生长抑素样物质不是通过二硫键交换导致激素聚合产生的。在对下丘脑提取物进行分级分离时，与该物质共洗脱的蛋白水解酶实现了对15000较高M(r)形式生长抑素的体外加工。在中性pH条件下，会产生中间较高M(r)形式以及M(r)为1600的生长抑素样物质。这种加工活性在酸性pH或酸/尿素介质中可能会受到强烈抑制，或者通过对15000较高M(r)形式的选择性免疫吸附而消除。胰蛋白酶和7S神经生长因子的γ亚基都不能产生这种加工，这表明可能涉及具有其他特异性的酶。得出的结论是，小鼠下丘脑生长抑素的生物合成可能通过一种较高M(r)的前体进行，该前体被加工成中间形式，进而产生十四肽激素。
