PHYSICOCHEMICAL CHARACTERIZATION OF THE NUCLEATIONAL CORE OF MATRIX VESICLES

While previous studies revealed that matrix vesicles (MV) contain a nu cleational core (NC) that converts to apatite when incubated with synt hetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca2+ and P-i-rich NC. MV, isolated from growth plate cartilage, w ere analyzed directly by solid-state P-31 NMR, or incubated with hydra zine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission ele ctron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamid e gel electrophoresis, We found that most of the MV proteins and lipid s could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis show ed that annexin V, a phosphatidylserine (PS)-dependent Ca2+-binding pr otein, was the major protein in the NC; high performance thin-layer ch romatographic analysis revealed that the detergents removed the majori ty of the polar lipids, but left significant free cholesterol and fatt y acids, and small but critical amounts of PS. Transmission electron m icroscopy showed that the NC was composed of clusters of similar to 1. 0 nm subunits, which energy-dispersive analysis with x-rays revealed c ontained Ca and P-i with a Ca/P ratio of 1.06 +/- 0.01, Electron diffr action, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline. H-1-Cross-polarization P-31 MMR indicated that the solid phase of MV was an HPO42--rich mixture o f amorphous calcium phosphate and a complex of PS, Ca2+, and P-i, Take n together, our findings indicate that the NC of MV is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2 +-P-i, annexin V, and other proteins and lipids.