Abstract
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+, Pi, and Zn2+. Extraction with 1 M NaCl removed much of the Ca2+ and Pi present in MV, markedly reducing Ca2+ accumulation; these effects could be prevented by low levels of Ca2+ and Pi 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 Pi, 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+, Pi, phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca2+ and Pi 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.
Highlights
Alization; further treatment with detergents tboypass [6] and have been shown to be widely distributed in mineralthe membrane reactivated matrix vesicles (MV) mineralization
Formation of Ca2+:acidic anthroline complexed Zn2+and stimulated mineraliza- phospholipidPi complexes, which has been shown to occur tion;addition of Zn2+ tosyntheticcartilagelymph within the cell before MV formation [6], is thought to trigger blocked the ability of the core to mineralize. These vesiculation [11].Complex formation is believed to bemestudies show that once the nucleational core complex diated by influx of Ca2+into the periphery of Pi-rich cells is formed, themembrane-enclosed domainis no longer [12], forming amorphous calcium phosphate that becomes essential for MV calcification
Effect of Detergent Treatment on Membrane Integrity-To assess the integrity of the vesicle membrane after extraction with TX-100, lactate dehydrogenase (LDH) activities were measured in the supernatant and pellet fractions. (LDH is a latent enzyme in MV present in the vesicle lumen [31].)Our findings showed that treatment of CRMV with TX-100 above the critical micelle concentration (CMC) caused the vesicle membrane to become leaky, and large amounts of LDH activity were released into the supernatantof the detergent extract
Summary
’The abbreviations used are: MV, matrix vesicles; CRMV, collaof calcium phosphate mineral crystal formation in vertebrate genase-released MV, SCL, synthetic cartilage lymph; LDH, lactate dehydrogenase; ACP, amorphous calcium phosphate; OCP, octacal-. CRMV (40 pg of protein) were treated with various detergents, and the resulting pellets after centrifugation (Beckman Airfuge, 22 psi, 130,000 X g, 7 min) were resuspended in SCL. Tissues were digested 0.1 ml was used for analysis of total P as Pi. For Pi and Ca analysis, at 37 "C in 0.1%trypsin (type111,Sigma) in syntheticcartilage lymph the 0.1 N HCl extract of CRMV wasclarified by centrifugation using (SCL) for 20 min. Gel Electrophoresis and Western Blot Analyses-SDS-PAGE was used to analyze CRMV and CRMV extracts after the various treatwere resuspended in SCL for ' T a uptake studies. The pellet was extractions was analyzed by immunoblotting with the appropriate incubated with water for 20 min at 25 "C.The treated pellet was monospecific rabbit antibodies to these avian proteins. The bands wereviewed by spraying with cupric-phosphoric acid charring reagent (10% CuS04 in 8% H3P04) and heating at 180 "C for 20 min [30]
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