Lepage-Lezin A, Joseph-Bravo P, Devilliers G, Benedetti L, Launay J M, Gomez S, Cohen P
Groupe de Neurobiochimie Cellularie et Molécularie de l'Université Pierre et Marie Curie, Unité de Recherches Associée 554 du Centre National de la Recherche Scientifique, Paris, France.
J Biol Chem. 1991 Jan 25;266(3):1679-88.
Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.
生长抑素前体的蛋白水解加工产生了几种肽，包括生长抑素-14（S-14）、生长抑素-28（S-28）和生长抑素-28（1-12）（S-28(1-12)）。通过对大鼠皮质或下丘脑细胞生物合成分泌装置的富集组分中的这些产物进行定量评估，确定了这些切割发生的亚细胞位点。通过不连续梯度离心获得每个主要细胞区室，并通过特异性酶标记物和电子显微镜进行表征。在色谱分离后，通过放射免疫测定法测量前生长抑素衍生的片段。使用了两种特异性抗体，分别用于鉴定由单碱性（精氨酸）和双碱性（精氨酸-赖氨酸）切割位点处的肽键水解产生的S-28(1-12)或S-14。这些抗体还揭示了在前体生长抑素衍生形式的COOH末端含有相应的十二肽和十四肽序列。虽然富含内质网的组分中前生长抑素水平最高，但在高尔基体中前体的比例明显较低。在后一组分中，还存在其他加工形式，即S-14和S-28(1-12)以及前生长抑素（pro-S(1-76)）的NH2末端结构域（1-76）。莫能菌素或在低温下预孵育对细胞内运输的抑制导致富含高尔基体的组分中前生长抑素衍生肽增加。最后，使用针对S-28(1-12)和S-14表位产生的抗体进行免疫金标记，结果表明这些形式几乎只存在于富含高尔基体的组分中，主要位于囊泡和小泡表面。这些数据共同表明，在大鼠神经细胞中，单碱性和双碱性位点的前生长抑素蛋白水解加工在高尔基体水平开始。
