Purification of a cartilage-derived growth factor

A growth factor was isolated from bovine cartilage and purified by a combination of gel filtration chromatography and isoelectric focusing. In the first step of the purification, growth factor activity as measured by stimulation of DNA synthesis in quiescent 3T3 cells or chondrocytes, was extracted by incubation of cartilage with 1 M guanidine hydrochloride, pH 6.0. The crude cartilage extract was analyzed by gel filtration on a column equilibrated with 4 M guanidine hydrochloride and 5 mM dithiothreitol. All of the cartilage-derived growth factor activity migrated in a region corresponding to molecular weights between 12,000 and 20,000. The low molecular weight fraction was analyzed by isoelectric focusing in the presence of 6 M urea. Two major active fractions were found, with isoelectric points of approximately 9 and 10. Purification to homogeneity was accomplished by several cycles of gel filtration of the pI 9 fraction on columns equilibrated with 4 M guanidine hydrochloride and 5 mM dithiothreitol. The purified growth factor migrated as a single polypeptide band in two polyacrylamide gel electrophoresis systems, sodium dodecyl sulfate polyacrylamide gel electrophoresis and acid-urea polyacrylamide gel electrophoresis. The molecular weight of the cartilage-derived growth factor was estimated to be between 16,000 and 18,000 by gel filtration and 16,400 by SDS polyacrylamide gel electrophoresis. Gel filtration chromatography of the pI 10 fraction also yielded an active purified 16,400 molecular weight growth factor. Approximately 1 to 2.5 micrograms/ml of purified cartilage-derived growth factor was necessary to half-maximally stimulate DNA synthesis in 3T3 cells and chondrocytes.