Nomata Y, Watanabe T, Wada H
J Biochem. 1983 Oct;94(4):1269-78. doi: 10.1093/oxfordjournals.jbchem.a134472.
In a continuation of previous work [Nomata et al. (1983) J. Biochem. 93, 825-831], this paper reports the purification and properties of the two other acidic proteins from human brain, and the immunochemical comparison of the three proteins. The protein described previously was named Glu-50 protein, because 50% of its amino acid residues were glutamic acid. By analogy, the proteins reported here were named Glu-20 and Glu-35 proteins on the basis of their glutamic acid contents. Both proteins were purified to a homogeneous state from human brain by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and gel filtration on Sephadex G-75 and G-100, like Glu-50 protein. The molecular weights of the Glu-20 and Glu-35 proteins in the native state were 61,000 and 48,000 daltons and the values in SDS medium were 60,000 and 16,000, respectively, suggesting that Glu-20 protein is a monomer, and Glu-35 a trimer. The isoelectric points of Glu-20 and Glu-35 proteins were pH 4.2 and 3.6, respectively. Glu-20 protein contained 20.5% Glu, 18.4% Asp and 10.5% Lys, and Glu-35 protein contained 33.9% Glu and 17.2% Asp. The N-terminal amino acids of both proteins were Gly, whereas the C-termini of Glu-20 and Glu-35 proteins were Leu and Lys, respectively. Glu-20 protein gave a slightly positive periodic acid-Schiff reaction, suggesting that it is a glycoprotein, but Glu-35 protein gave a negative reaction. The three acidic proteins, Glu-50, Glu-35, and Glu-20 proteins, were compared immunochemically. Antibodies could be produced in rabbits against Glu-50 and Glu-20 proteins only after immunization with the performate-oxidized proteins, but no antibody could be produced against Glu-35 by similar immunization. Anti Glu-50 antibody reacted only with Glu-50 protein, not with Glu-35 or Glu-20 protein, calmodulin, or S-100 protein. Similarly, anti Glu-20 antibody was specific for Glu-20 protein and did not cross-react with Glu-35 or Glu-50 protein, calmodulin, or S-100. Anti S-100 antibody did not react with Glu-50, Glu-35, or Glu-20 protein. The cross-reactivities of these antibodies were tested with extracts of various tissues of a goat. Anti Glu-50 antibody reacted only with an extract of the brain, not with extracts of other tissues, whereas anti Glu-20 antibody reacted with all the tissue extracts examined.
作为之前工作[野间田等人（1983年），《生物化学杂志》93卷，825 - 831页]的延续，本文报道了从人脑中分离出的另外两种酸性蛋白质的纯化及特性，以及这三种蛋白质的免疫化学比较。先前描述的蛋白质被命名为Glu - 50蛋白，因为其氨基酸残基的50%为谷氨酸。以此类推，这里报道的蛋白质根据其谷氨酸含量分别命名为Glu - 20和Glu - 35蛋白。与Glu - 50蛋白一样，通过硫酸铵分级沉淀、DEAE - 葡聚糖A - 50柱色谱以及在葡聚糖G - 75和G - 100上的凝胶过滤，从人脑中纯化得到这两种蛋白质至均一状态。天然状态下Glu - 20和Glu - 35蛋白的分子量分别为61,000和48,000道尔顿，在SDS介质中的值分别为60,000和16,000，这表明Glu - 20蛋白是单体，而Glu - 35是三聚体。Glu - 20和Glu - 35蛋白的等电点分别为pH 4.2和3.6。Glu - 20蛋白含有20.5%的谷氨酸、18.4%的天冬氨酸和10.5%的赖氨酸，Glu - 35蛋白含有33.9%的谷氨酸和17.2%的天冬氨酸。两种蛋白质的N端氨基酸均为甘氨酸，而Glu - 20和Glu - 35蛋白的C端分别为亮氨酸和赖氨酸。Glu - 20蛋白对高碘酸 - 席夫反应呈轻微阳性，表明它是一种糖蛋白，而Glu - 35蛋白呈阴性反应。对三种酸性蛋白Glu - 50、Glu - 35和Glu - 20蛋白进行了免疫化学比较。仅在用过甲酸氧化的蛋白质免疫后，兔子才能产生针对Glu - 50和Glu - 20蛋白的抗体，但通过类似免疫不能产生针对Glu - 35的抗体。抗Glu - 50抗体仅与Glu - 50蛋白反应，不与Glu - 35或Glu - 20蛋白、钙调蛋白或S - 100蛋白反应。同样，抗Glu - 20抗体对Glu - 20蛋白具有特异性，不与Glu - 35或Glu - 50蛋白、钙调蛋白或S - 100发生交叉反应。抗S - 100抗体不与Glu - 50、Glu - 35或Glu - 20蛋白反应。用山羊各种组织的提取物测试了这些抗体的交叉反应性。抗Glu - 50抗体仅与脑提取物反应，不与其他组织的提取物反应，而抗Glu - 20抗体与所有检测的组织提取物反应。
