Huang J S, Huang S S, Kuo M D
J Biol Chem. 1986 Sep 5;261(25):11600-7.
A large-scale purification procedure for brain-derived growth factors, without chromatography in 0.1% trifluoracetic acid, has been developed. Two related brain-derived growth factors have been purified to homogeneity from bovine brain using this procedure involving ammonium sulfate fractionation, followed by chromatography on CM-Sephadex C-50, sulfated Sephadex G-50, and heparin-Sepharose 4B. Brain-derived growth factor A (BDGF-A, molecular weight approximately 16,000) and brain-derived growth factor B (BDGF-B, molecular weight approximately 17,000) were eluted from heparin-Sepharose 4B at 1.2 and 1.6 M NaCl, respectively. Both BDGF-A and BDGF-B have a pI value of 5.7, have the same specific mitogenic activity, and react with mouse anti-BDGF-A antiserum. Both growth factors have a broad spectrum of mitogenic activity (vascular and aorta endothelial cells, chondrocytes, osteoblasts, epithelial cells, glial cells, fibroblasts, and smooth muscle cells), with a half-maximum effect at 10-20 pM. The binding of BDGF to bovine aorta endothelial cells and Swiss mouse 3T3 cells has been characterized. Binding of 125I-BDGF-A to these cells reached equilibrium in less than 15 min. Scatchard plot analysis of the binding of 125I-BDGF-A to endothelial cells and 3T3 cells showed a single class of high-affinity receptor with Kd values of 20 +/- 5 and 13 +/- 3 pM and receptor numbers/cell of 7,000 +/- 1,000 and 20,000 +/- 3,000, respectively. BDGF-B competed for 125I-BDGF-A binding in the same manner as unlabeled BDGF-A, suggesting that BDGF-A and BDGF-B bind to the same receptor. Basic pituitary fibroblast growth factor appeared to be a weak inhibitor, whereas platelet-derived growth factor and epidermal growth factor had no effect on 125I-BDGF-A binding. Protamine, histone, polylysine, and polyarginine are potent inhibitors of the mitogenic activity of BDGF-A and BDGF-B and of binding of 125I-BDGF-A to responsive cells. The half-time (t1/2) of internalization and degradation of cell surface-bound 125I-BDGF is 3 h.
已开发出一种用于脑源性生长因子的大规模纯化程序，该程序在0.1%三氟乙酸中无需色谱法。使用该程序，通过硫酸铵分级分离，随后在CM-葡聚糖凝胶C-50、硫酸化葡聚糖凝胶G-50和肝素-琼脂糖4B上进行色谱分离，从牛脑中纯化出了两种相关的脑源性生长因子，并达到了均一性。脑源性生长因子A（BDGF-A，分子量约为16,000）和脑源性生长因子B（BDGF-B，分子量约为17,000）分别在1.2和1.6 M NaCl浓度下从肝素-琼脂糖4B上洗脱下来。BDGF-A和BDGF-B的pI值均为5.7，具有相同的特异性促有丝分裂活性，并与小鼠抗BDGF-A抗血清发生反应。两种生长因子都具有广泛的促有丝分裂活性（血管和主动脉内皮细胞、软骨细胞、成骨细胞、上皮细胞、神经胶质细胞、成纤维细胞和平滑肌细胞），在10 - 20 pM时达到最大效应的一半。已对BDGF与牛主动脉内皮细胞和瑞士小鼠3T3细胞的结合进行了表征。125I-BDGF-A与这些细胞的结合在不到15分钟内达到平衡。对125I-BDGF-A与内皮细胞和3T3细胞结合的Scatchard图分析显示，存在一类单一的高亲和力受体，其Kd值分别为20±5和13±3 pM，受体数量/细胞分别为7,000±1,000和20,000±3,000。BDGF-B与未标记的BDGF-A以相同方式竞争125I-BDGF-A的结合，表明BDGF-A和BDGF-B结合到相同的受体上。碱性垂体成纤维细胞生长因子似乎是一种弱抑制剂，而血小板衍生生长因子和表皮生长因子对125I-BDGF-A的结合没有影响。鱼精蛋白、组蛋白、聚赖氨酸和聚精氨酸是BDGF-A和BDGF-B促有丝分裂活性以及125I-BDGF-A与反应性细胞结合的有效抑制剂。细胞表面结合的125I-BDGF内化和降解的半衰期（t1/2）为3小时。
