Lny. Wu et al., CHARACTERIZATION OF THE NUCLEATIONAL CORE COMPLEX RESPONSIBLE FOR MINERAL INDUCTION BY GROWTH-PLATE CARTILAGE MATRIX VESICLES, The Journal of biological chemistry, 268(33), 1993, pp. 25084-25094
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.