Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles.
Uptake of mineral ions by isolated matrix vesicles (MV) incubated in synthetic cartilage lymph follows a consistent pattern. After an initial lag period, MV rapidly accumulate large amounts of Ca2+ and Pi before the appearance of crystalline mineral. The ability of MV to accumulate Ca2+ is readily destroyed by proteases, indicating that proteins are important in Ca2+ accumulation. Since MV contain significant amounts of phosphatidylserine (PS), an acidic phospholipid with affinity for Ca2+, it seemed probable that this lipid might also contribute to Ca2+ binding. The development of methods for reproducible isolation of pure active MV enabled us to search for factors responsible for the rapid accumulation of Ca2+. Reported here are studies which reveal that a set of intensely staining MV proteins, extractable with EGTA, selectively bind to Ca2+, but only in the presence of acidic phospholipids. These 30-36-kDa proteins form readily sedimentable insoluble ternary complexes of protein, Ca2+, and lipid in the presence of low levels of Ca2+. With liposomes composed of PS, alone or in combination with phosphatidylethanolamine, submicromolar levels of Ca2+ or certain other divalent cations, but not Mg2+, are sufficient to form the complexes. The physical and chemical properties of these MV proteins appear to be like those of the calpactin family of membrane-associated proteins. In fact, these MV proteins were found to cross-react with antibodies to calpactin II. Thus, calpactins appear to be important protein constituents of avian growth plate MV. This finding helps explain the enrichment in PS previously noted in MV and may also point to the mechanism by which MV rapidly accumulate Ca2+.
Genge
B R
BR
Department of Chemistry, University of South Carolina, Columbia 29208.
Wu
L N
LN
Wuthier
R E
RE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D002645:Chickens; D004533:Egtazic Acid / Q000494:pharmacology; D006132:Growth Plate / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D008081:Liposomes; D010743:Phospholipids; D004533:Egtazic Acid; D002118:Calcium