Purification of water-soluble bone-inductive protein from bovine demineralized bone matrix.
The water-soluble fraction containing bone-inductive activity was purified from guanidine-hydrochloride extracts of bovine demineralized bone. The purification steps include ultrafiltration, dialysis, affinity chromatography on heparin-Sepharose and gel chromatography on Sephacryl S-200. Combination of these steps was proven to be an effective and rapid method for the purification of this protein. Subcutaneous implantation of the water-soluble protein with type I collagen was carried out in the thorax of rats. When alkaline phosphatase activity and calcium content in implants were used as indices for purification, the water-soluble bone-inductive protein was purified > 600-fold according to the enzyme activity and 64-fold according to the calcium content. A morphological examination revealed that many chondrocyte and osteoblast cells were seen in the location of the implanted material. Sodium dodecyl sulfate/gel electrophoresis of the protein produced in this way under non-reducing conditions revealed four protein bands of 18, 16, 14 and 11 kDa. None of the separated bands had any biological activity. This result suggests that the water-soluble bone-inductive activity depends on an associated form of various proteins in the range of 18 to 11 kDa.
Yoshimura
Y
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, University of Tokushima, Japan.
Hirano
A
A
Nishida
M
M
Kawada
J
J
Horisaka
Y
Y
Okamoto
Y
Y
Matsumoto
N
N
Yamashita
K
K
Takagi
T
T
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001846:Bone Development / Q000187:drug effects; D001857:Bone Matrix / Q000737:chemistry*; D019485:Bone Morphogenetic Proteins; D001842:Bone and Bones / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014462:Ultrafiltration
D019485:Bone Morphogenetic Proteins; D011506:Proteins; D000469:Alkaline Phosphatase; D002118:Calcium