Platelet-derived growth factor. I. High yield purification and evidence for multiple forms.
Platelet-derived growth factor has been purified from human outdated platelet-rich plasma with a 21% overall yield. This five-step procedure represents a 500,000-fold purification over serum. 6.4 ng/ml (2 X 10(-10) M) of purified platelet-derived growth factor stimulated DNA synthesis in quiescent, density arrested cultures of Swiss 3T3 cells to a level equivalent to that produced by 5% calf serum. The growth-promoting activity and specific binding to 3T3 cells has been shown to be associated with four entities of molecular weights: 31,000, 29,000, 28,500, and 27,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In the presence of 2-mercaptoethanol, the four molecular species are converted to three inactive chains of lower molecular weights: 17,500, 16,000, and 14,400. Other methods of chemically cleaving the disulfide bonds were investigated: reductive cleavage by S-sulfonation, reductive cleavage with dithiothreitol, and performic acid oxidation. In all cases, mitogenic activity was reduced by 80-100%. Attempts to restore mitogenic activity after reduction were unsuccessful. Two-dimensional 125I-peptide mapping of the nonreduced and reduced multiple forms was also investigated. The four nonreduced moieties gave basically identical maps. The maps of the reduced 17,500- and 16,000-dalton chains were also essentially identical, but the map of the reduced 14,400-dalton chain was significantly different. We propose that platelet-derived growth factor consists of two polypeptide chains: a 14,400-dalton chain and either a 17,500- or a 16,000-dalton chain.
D000818:Animals; D001792:Blood Platelets / Q000032:analysis*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D008934:Mitogens / Q000302:isolation & purification*; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor
D006133:Growth Substances; D008934:Mitogens; D010455:Peptides; D010982:Platelet-Derived Growth Factor