Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.

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Abstract
Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent MW of 18,500 .+-. 500 and represents < 0.001% of the wet weight of bone tissue. A MW 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A MW 24,000 protein and about half of a MW 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a MW 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the MW 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent MW 18,500, 17,500 and 17,000 are eluted at 0.10, 0.15 and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The MW 18,500 protein has the amino acid composition of acidic polypeptide and includes 4 half-cystine residues; the pI [isoelectric point] is 4.9-5.1. The MW 22,000 component is a chromoprotein resembling ferritin. The NH2-terminal amino acid sequence of the MW 17,500 protein simulates histone H2B. The MW 17,000 protein may possess calmodulin activity. Aggregates of the MW 18,500 and other proteins induce formation of large deposits of bone; the MW 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.