Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes, phospholipids, calcium and phosphate

Watkins, E.L.; Stillo, Joseph V.; Wuthier, Roy E.

doi:10.1016/0304-4165(80)90303-7

Cited by 49 publications

(18 citation statements)

References 27 publications

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“…The role of NTPPPH activity in physiologic endochondral mineralization is reinforced by the presence of NTPPPH in growth plate cartilage matrix vesicles (11)(12)(13)(14)(15). NTPPPH activity is also linked to pathologic mineralization of adult hyaline articular cartilage, i.e., deposition of crystals of hydroxyapatite and other basic calcium phosphates (16), and of calcium pyrophosphate dihydrate (CPPD) (in CPPD crystal deposition disease, or chondrocalcinosis) (17)(18)(19)(20).…”

Section: Introductionmentioning

confidence: 99%

Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.

Huang

Rosenbach²,

Vaughn³

et al. 1994

J. Clin. Invest.

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Section: Introductionmentioning

confidence: 99%

Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.

Huang

Rosenbach²,

Vaughn³

et al. 1994

J. Clin. Invest.

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“…MV are produced by budding from the plasma membrane into sites of intended calcification by cellularly controlled mechanisms (4 -6). Previous biochemical (7)(8)(9) and ultrastructural (10,11) studies have shown that MV contain large amounts of calcium and phosphate; however, the initial mineral phase has not yet been characterized. X-ray diffraction (12), and FT-IR (13) and FT-Raman (14) spectroscopic analyses all indicate that the initial mineral form in MV is noncrystalline.…”

Section: ؉mentioning

confidence: 99%

Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

Genge

Dunkelberger

et al. 1997

Journal of Biological Chemistry

139

154

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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 Ca 2؉ and P i -rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solidstate 31 P 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 Ca 2؉ -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 ϳ1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and P i 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.

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“…However, to the best of our knowledge, these two types of vesicles have not been reported to occur in bone. Moreover, Watkins et al [22] have recently demonstrated that fractionation of cartilage revealed vesicle preparations with acid phosphatase activity. This activity was 20-fold lower than the activity of alkaline phosphatase.…”

Section: Edvfamentioning

confidence: 98%

Correlative morphometric and biochemical analysis of purified extracellular matrix vesicles from rat alveolar bone

Deutsch

Schwartz

et al. 1983

Calcif Tissue Int

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Extracellular matrix vesicles were isolated from rat alveolar bone and purified by either gel filtration or discontinuous sucrose density gradient. Morphometric evaluation of electron-micrographs of pellets of purified and nonpurified vesicle fractions was correlated with the activity of vesicular enzymes. A high correlation was found between the percentage of area occupied by vesicles with electron-dense content (electron-dense vesicle fractional area) and the enzymatic activity. Highest enzymatic specific activities and electron-dense vesicle fractional area were recorded in the "light" vesicle-enriched fraction obtained after equilibrium density centrifugation. These parameters revealed lowest values in the "heavy" vesicle-enriched fraction resolved by the same methods. The combined electron-dense and electron-lucent fractional area (vesicular fractional area) was similar in the different purified fractions. It is therefore suggested that the fraction obtained by gel filtration contains both "light" and "heavy" vesicles. Morphometric study is proposed as an additional criterion for the degree of purification of matrix vesicle preparations.

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Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes, phospholipids, calcium and phosphate

Cited by 49 publications

References 27 publications

Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.

Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.

Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

Correlative morphometric and biochemical analysis of purified extracellular matrix vesicles from rat alveolar bone

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