Liu K P, Gershon M D, Tamir H
J Neurochem. 1985 Apr;44(4):1289-301. doi: 10.1111/j.1471-4159.1985.tb08756.x.
Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K from of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by--SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants--56K form showed higher affinity for 5-HT (KD1 = 0.4 nM; KD2 = 32 nM), whereas the 45K form showed lower affinity (KD1 = 9.7 nM; KD2 = 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity--56K (19:1), 45K (10:1); (3) isoelectric point--the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)
血清素结合蛋白（SBP）存在于哺乳动物中枢和外周血清素能神经元的突触小泡中。5-羟色胺（5-HT，血清素）在体内作为与SBP的复合物被生理性储存。已检测到两种形式的SBP，其表观分子量分别为45,000和56,000（45K和56K）。为了更全面地了解这两种形式之间的关系，我们将这两种蛋白质纯化至同质并对其进行了部分特性分析。纯化步骤包括硫酸铵分级分离以及在琼脂糖4 - B、亲和凝胶蓝、羟基磷灰石和磷酸纤维素上进行色谱分离。通过这些方法，45K形式的SBP被获得纯品，而56K形式的SBP纯度约为90%。为了分离用于诱导抗体的纯56K SBP，该蛋白质通过十二烷基硫酸钠 - 凝胶电泳随后进行电洗脱进一步纯化。56K形式的SBP因此被分离出来，但处于变性状态；其纯度通过二维凝胶电泳确定。两种形式的SBP（纯45K和90%纯未变性56K SBP）在5-HT结合能力、Fe2 +对5-HT结合的增强作用以及被 - SH试剂、螯合剂和钠盐抑制方面相似。针对纯56K形式的SBP产生的抗体与45K SBP发生交叉反应。两种形式的SBP在以下特性方面存在差异：（1）解离常数——56K形式对5-HT显示出更高的亲和力（KD1 = 0.4 nM；KD2 = 32 nM），而45K形式显示出较低的亲和力（KD1 = 9.7 nM；KD2 = 120 nM）；（2）低亲和力与高亲和力5-HT结合位点的数量比——56K（19:1），45K（10:1）；（3）等电点——56K形式的SBP比45K形式（6.1）更偏酸性（5.6和5.9）；（4）神经节苷脂和碳酸氢盐对结合的增强作用。为了确定45K形式的SBP是在体内存在还是在分离过程中由蛋白水解产生，又进行了另外两个实验。（1）我们在匀浆缓冲液中添加了蛋白酶抑制剂混合物；这种添加并没有改变两种形式SBP的比例。（2）我们将富含45K形式SBP的中枢神经系统区域（脊髓）与富含56K形式SBP的区域（中缝核）混合并一起匀浆。同样，通过聚丙烯酰胺凝胶电泳判断，该过程未能改变两种形式SBP的比例。（摘要截短于400字）
