Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles

The factors that drive mineralization of matrix vesicles (MV) have proven difficult to elucidate; in the present studies, various detergent, chemical, and enzyme treatments were used to reveal the nature of the nucleational core. Incubation with detergents that permeabilized the membrane enhanced calcification of treated MV incubated in synthetic cartilage lymph. While detergents removed most of the membrane lipid, they left significant amounts of the MV annexins and nearly all of the Ca²⁺, P(i), and Zn²⁺. Extraction with 1 M NaCl removed much of the Ca²⁺ and P(i) present in MV, markedly reducing Ca²⁺ accumulation; these effects could be prevented by low levels of Ca²⁺ and P(i) in the NaCl extractant. Treatment with chymotrypsin appeared to damage proteins required for MV mineralization; further treatment with detergents to bypass the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca²⁺ and P(i), destroying their ability to mineralize; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o- phenanthroline complexed Zn²⁺ and stimulated mineralization; addition of Zn²⁺ to synthetic cartilage lymph blocked the ability of the core to mineralize. These studies show that once the nucleational core complex is formed, the membrane-enclosed domain is no longer essential for MV calcification. Our findings indicate that the MV core contains two main components as follows: a smaller membrane-associated complex of Ca²⁺, P(i), phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca²⁺ and P(i) bound to lumenal proteins. These proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystalline phase. Once nucleated, the crystalline phase appears to feed on protein- bound mineral ions until external ions enter through the MV ion channels. Zn²⁺ appears to regulate gating of the ion channels and conversion of the nucleational complex to the crystalline state.