Tamir H, Liu K P, Hsiung S, Adlersberg M, Gershon M D
Division of Neuroscience, New York State Psychiatric Institute, NY 10032.
J Neurochem. 1994 Jul;63(1):97-107. doi: 10.1046/j.1471-4159.1994.63010097.x.
Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5-HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5-HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56- and 45-kDa forms of SBP are produced by posttranslational cleavage of a 68-kDa precursor molecule; and (b) that 45-kDa SBP is a constituent of serotonergic secretory vesicles. Pulse-chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with L-[35S]methionine, the cells were incubated in the presence of an excess of unlabeled L-methionine for 0, 30, 60, or 90 min at 37 degrees C. Alternatively, the chase was performed under conditions (20 degrees C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20 degrees C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post-Golgi compartment. When secretion was induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was released to the medium. When antibodies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement.(ABSTRACT TRUNCATED AT 400 WORDS)
血清素结合蛋白（SBP）存在于所有储存血清素（5-HT）的神经外胚层来源细胞中。已检测到三种形式的SBP（68、56和45 kDa），且干扰5-HT结合的SBP抗体与这些蛋白质中的每一种都发生反应。当前实验检验了两个假设：（a）56 kDa和45 kDa形式的SBP是由68 kDa前体分子的翻译后切割产生的；（b）45 kDa SBP是血清素能分泌囊泡的一个组成部分。以甲状腺髓样癌细胞为模型进行了脉冲追踪实验。这些神经外胚层来源的细胞产生5-HT和所有三种形式的SBP。用L-[35S]甲硫氨酸脉冲标记20分钟后，将细胞在过量未标记的L-甲硫氨酸存在下于37℃孵育0、30、60或90分钟。或者，在延迟或阻止新合成蛋白质从粗面内质网通过高尔基体转运的条件下（20℃，抑制ATP生成）进行追踪。孵育后，洗涤并溶解细胞，免疫沉淀SBP。免疫沉淀物中的放射性蛋白质通过电泳分离并定量。脉冲后立即发现三种形式的SBP均被35S标记。在每个追踪间隔，35S标记的68 kDa、56 kDa和45 kDa SBP的相对比例保持不变。当在20℃或阻断ATP生物合成的条件下进行追踪时，这些比例没有变化。这些观察结果表明，每种形式的SBP都是翻译的初级产物，较小形式的SBP不是由较大分子切割产生的，并且翻译初级产物的大小不会因进入高尔基体或高尔基体后区室而改变。当诱导分泌时，45 kDa SBP被释放到培养基中，而56 kDa或68 kDa SBP则未被释放。当在诱导分泌时向培养基中加入45 kDa SBP的抗体时，抗体结合位点在细胞表面呈现为斑块状。由于这些位点，当细胞在存在45 kDa SBP抗体和豚鼠补体的情况下被刺激分泌时会发生裂解。（摘要截短至400字）
