Summary
Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm3 band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm3 band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm3 band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm3 band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm3 fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation withβ-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.
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Kahn, S.E., Jafri, A.M., Lewis, N.J. et al. Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage. Calc. Tis Res. 25, 85–92 (1978). https://doi.org/10.1007/BF02010755
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DOI: https://doi.org/10.1007/BF02010755