Isolation and characterization of native adult osteonectin.
Noncollagenous bovine bone proteins were obtained from EDTA-solubilized extracts of adult bovine bone in the absence of denaturants. Native osteonectin was isolated from the noncollagenous bone proteins by ion-exchange chromatography using DEAE-Sephadex A-50 and DEAE-Sephadex A-25, followed by gel filtration on Sephadex G-100. Comparison of the physical and chemical properties (i.e. electrophoric mobility, amino acid composition and pI) of this protein with those reported by Termine et al. (Termine, J.D., Belcourt, A.B., Conn, K.M., and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408) indicate that this protein is osteonectin. Sedimentation equilibrium analyses in the presence of 6 M guandinium chloride, 10 mM Ca2+, 10 mM EDTA, or 0.15 M NaCl all yielded a molecular weight of 29,100 +/- 900. 125I-Osteonectin underwent saturable and exchangeable binding to hydroxyapatite and calf skin collagen. Eleven mg of 125I-osteonectin bound to 1.0 g of hydroxyapatite with a Kd of 8 X 10(-8) M. The intrinsic fluorescence of bovine osteonectin was partially quenched when micromolar Ca2+ was added, indicating a high affinity Ca2+ interaction. Native osteonectin was found to reduce the rate of hydroxyapatite crystal seeded growth by 50% (1 IU) at a concentration of 1.6 X 10(-7) M at pH 7.4, 37 degrees C in 0.15 M NaCl. This makes osteonectin one of the most potent inhibitors of hydroxyapatite formation presently known and more than 5 times as effective as bone Gla protein (1 IU = 8 X 10(-7) M).
Romberg
R W
RW
Werness
P G
PG
Lollar
P
P
Riggs
B L
BL
Mann
K G
KG
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism; D003460:Crystallization; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D008433:Mathematics; D015676:Osteonectin
D000596:Amino Acids; D002352:Carrier Proteins; D006882:Hydroxyapatites; D015676:Osteonectin; D003094:Collagen; D017886:Durapatite