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 pro ven 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 c artilage lymph. While detergents removed most of the membrane lipid, t hey 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 e ffects could be prevented by low levels of Ca2+ and P(i) in the NaCl e xtractant. Treatment with chymotrypsin appeared to damage proteins req uired for MV mineralization; further treatment with detergents to bypa ss the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca2+ and P(i), destroying their ability to minerali ze; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o-phenanthroline compl exed 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 me mbrane-enclosed domain is no longer essential for MV calcification. Ou r findings indicate that the MV core contains two main components as f ollows: a smaller membrane-associated complex of Ca2+, P(i), phosphati dylserine, and the annexins that nucleates crystalline mineral formati on, and a larger pool of Ca2+ and P(i) bound to lumenal proteins. Thes e proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystal line phase. Once nucleated, the crystalline phase appears to feed on p rotein-bound mineral ions until external ions enter through the MV ion channels. Zn2+ appears to regulate gating of the ion channels and con version of the nucleational complex to the crystalline state.