Evidence for the formation of a complex between osteopontin and osteocalcin.
We hypothesize that the mechanisms governing bone formation and remodeling involve the assembly of some of the components of the extracellular matrix into supramolecular complexes. We have examined the associations of osteopontin (OPN) with other proteins isolated from demineralized rat long bones. Three ligand binding techniques were used to demonstrate the formation of complexes between osteopontin and osteocalcin (OCN). Using gel overlay assays, the binding between soluble 125I-OPN and OCN immobilized in acrylamide gels was visualized. Competition for 125I-OPN-OCN complexes was demonstrated when unlabeled OCN-enriched bone extract was included in gel overlay solutions. Also, gel overlay assays showed 125I-OCN binding to OPN. Saturable binding was shown in solid-phase filter binding assays, which yielded an equilibrium binding constant of moderately high affinity (approximately 10(-8) M). Specificity of OPN-OCN complex formation was confirmed by measuring binding in the presence of unlabeled OPN and OCN versus a bone-localized serum protein, alpha 2HS-glycoprotein. Finally, the formation of soluble complexes were demonstrated in a modified Hummel-Dreyer gel filtration assay. These results indicate that OPN and OCN form complexes in vitro. The possible functions of OPN-OCN complexes in osteoclast recruitment and attachment are discussed.
Ritter
N M
NM
Department of Biological Chemistry, University of Texas Health Science Center, Houston.
Farach-Carson
M C
MC
Butler
W T
WT
D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015675:Osteocalcin / Q000737:chemistry / Q000378:metabolism*; D053495:Osteopontin; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D015675:Osteocalcin; D053495:Osteopontin