High-yield purification of platelet-derived endothelial cell growth factor: structural characterization and establishment of a specific antiserum.
Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa protein that stimulates the growth of endothelial cells [Miyazono, K., et al. (1987) J. Biol. Chem. 262, 4098-4103]. Here, we describe a method to purify large quantities of PD-ECGF from human platelet lysate at a high yield (14% overall recovery). The purification method involves five steps, using high-performance liquid chromatography grade hydroxylapatite and hydrophobic chromatographies as the two final steps. The purified material contained two major components of apparent molecular weight values of 46,000 and 44,000. These components coeluted in a high-resolving reversed-phase chromatography and were found to give similar peptide maps after treatments with staphylococcal V8 protease, suggesting that the 44-kDa form is related to the 46-kDa molecule. Partial tryptic digestion of native PD-ECGF revealed that the molecule contains a trypsin-resistant domain of 37-39 kDa. A rabbit antiserum was produced against the purified material and was found to specifically recognize PD-ECGF in immunoblotting. When added to the cell culture medium, an immunoglobulin fraction of the antiserum neutralized the activity of purified PD-ECGF. Furthermore, it completely neutralized the endothelial cell mitogenic activity of platelet lysate, indicating that PD-ECGF is the only mitogen in platelet lysate for this cell type.
Miyazono
K
K
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Heldin
C H
CH
D000818:Animals; D001792:Blood Platelets / Q000502:physiology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D006882:Hydroxyapatites; D007106:Immune Sera; D015151:Immunoblotting; D008970:Molecular Weight; D010449:Peptide Mapping; D010982:Platelet-Derived Growth Factor / Q000302:isolation & purification*; D013552:Swine
D006882:Hydroxyapatites; D007106:Immune Sera; D010982:Platelet-Derived Growth Factor; D017886:Durapatite