Amino Acid Sequence of Pulp Cell Growth Factor (PGF)

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The purification of pulp cell growth factor and dentin morphogenetic protein were performed, shich have the ability to stimulate proliferation of dental pulp cells and regulate differentiation into odontoblasts. Bovine demineralized dentin matrix was dissociatively extracted in 4 M guanidine HCl, and loaded onto hydroxyapatite affinity clumn. The eluate from the column in 100 mM potassium phosphate was concentrated and loaded agein onto herarin-Sepharose CL-6B column. The 0.5 M NaCl eluate dialyzed against distillled water and lyophilized was further purified by reversed-phase high pressure liquid chromatography using a linear gradient of 20 %-100 % acetonitrile. The column fractions 13, 14 and 15 (acetonitrile 48-69 %) had an inhibitory affect on alkaline phosphatase activity and fraction 14 had a stimulatory effect on DNA synthesis in primary pulp cell culture. Each fraction was applied for electrophoresis to cut into slices, and around 31-kDa and 25-kDa region proteins were eluted in extraphor elecrophoretic concentrator. Fraction 13-2 (31 kDa) had an inhibitory effect on alkaline phosphatase activity and fraction 14-4 (25 kDa) had a stimulatory effect on DNA synthesis. The 25-kDa protein was divided into half, 12.5 kDa and 31-KDa was not under reduecd condition, suggesting that these two proteins were not identical each other. We are now determining of amino acid sequences of these proteins.