Bovine brain-derived growth factor. Purification and characterization of its interaction with responsive cells.
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.
D000818:Animals; D001921:Brain / Q000502:physiology*; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004727:Endothelium / Q000166:cytology / Q000187:drug effects; D005260:Female; D005346:Fibroblast Growth Factors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor; D013936:Thymidine / Q000378:metabolism
D006133:Growth Substances; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D005346:Fibroblast Growth Factors; D013936:Thymidine