CALCIUM-BINDING PROPERTIES OF OSTEOPONTIN DERIVED FROM NONOSTEOGENIC SOURCES

Osteopontin (OP), purified from rat bone, binds Ca2+ but whether diffe rent molecular forms of OPs derived from non-osteogenic sources and no n-phosphorylated OP also possess this property remains to be determine d. Furthermore, it is not known which specific site or sites of the mo lecule bind Ca2+. In the present study, following an established proce dure, total proteins in the conditioned media from OP-synthesizing cel l cultures were separated by SDS-PAGE, transferred to Immobilon-P memb ranes, and incubated with (CaCl2)-Ca-45, then Ca2+ ions bound to prote in bands were analyzed by autoradiography. Purified OPs, and synthetic oligopeptides representing specific domains of the OP molecule were a dsorbed on the membrane and processed as described above. Our results show that OPs synthesized by normal rat kidney cells, oncogenically tr ansformed Rat-1 cells, OP purified from human milk, and non-phosphoryl ated OP secreted by 1(alpha),25-dihydroxyvitamin D-3-treated mouse epi dermal JB6 cells all bind detectable levels of Ca2+ with specificity. We also show that a synthetic peptide representing the domain of OP wh ich contains nine consecutive aspartic acid residues binds Ca2+ with s pecificity. It is probable, therefore, that a Ca2+-binding site reside s in this region of the OP molecule. We conclude that Ca2+-binding is a general property of OP, irrespective of its molecular mass and origi n, and the phosphate moieties of OP may not influence the conformation or accessibility of the Ca2+ affinity sites of the molecule.