Abstract
While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca2+ and Pi-rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solid-state 31P NMR, or incubated with hydrazine 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 electron 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-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca2+-binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of approximately 1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and Pi with a Ca/P ratio of 1.06 +/- 0. 01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline. 1H-Cross-polarization 31P NMR indicated that the solid phase of MV was an HPO42--rich mixture of amorphous calcium phosphate and a complex of PS, Ca2+, and Pi. Taken together, our findings indicate that the NC of MV is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2+-Pi, annexin V, and other proteins and lipids.
Highlights
While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized
Our findings indicate that the NC of MV is composed of an acidphosphate-rich amorphous calcium phosphate intermixed with PS-Ca2؉-Pi, annexin V, and other proteins and lipids
Chemical analyses revealed that the Ca and Pi content of the MV was relatively unchanged; ϳ98% of the Ca2ϩ and ϳ86% of the Pi were recovered in the NC
Summary
While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. We showed that it was possible to strip away nonessential components from the MV using various chemical and detergent extractions leaving a residual core that retained the ability to induce mineral formation [15] The activity of this detergent-stable nucleational core (NC) was inhibited by Zn2ϩ and was destroyed by treatment with pH 6 citrate buffer. Establishment of the physicochemical properties of the nucleational core (the first mineral phase) would provide an important clue to how MVs are formed and how they function as the primary nucleator in growth plate cartilage calcification In this investigation we examined the physicochemical properties of the residual core material in MV remaining after sequential treatments with enzyme, buffered salt, and detergent solutions. High performance thinlayer chromatography (HPTLC) was used to analyze lipids; SDS-PAGE was used to analyze proteins
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