tailieunhanh - Báo cáo khoa học: Chick embryo anchored alkaline phosphatase and mineralization process in vitro Influence of Ca 2+ and nature of substrates

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate,but containing calcium and GPI-bALP substrates,served asin vitro model of min-eral formation. The mineralization process was initiated by the formation of inorganic phosphate,arising from the hydrolysis of a substrate by GPI-bALP. Several minerali-zation media containing different substrates were analysed after an incubation time ranging from h to 144 h. | Eur. J. Biochem. 270 2 8S . 90 2003 FEBS 2003 doi Chick embryo anchored alkaline phosphatase and mineralization process in vitro Influence of Ca2 and nature of substrates Eva Hamade Gerard Azzar Jacqueline Radisson Rene Buchet and Bernard Roux Laboratoire de Physico-Chimie Biologique UMR CNRS 5013 Universite Claude Bernard Lyon I Villeurbanne France Bone alkaline phosphatase with glycolipid anchor GPI-bALP from chick embryo femurs in a medium without exogenous inorganic phosphate Uut contilining calcium and GPI-bALP substrates served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from h to 144 h. The measurements of Ca Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures while the nnalysis of X-ray i ira a ctiOTt CtUa allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine glucose 1 -phosphate glua 6-phosphate gUioo yy GPIALLP produced hydroxyapatite in a manner similar to that of b-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation then poorly formed hydroxyapatite crystalline structures were observed followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However hie hyd olysis of either ATP or ADP catals sed by GPI-bALP m caicium-containing medium did not lead to the formation of any hydroxyapatite crystals even after 144 h incubation time when hydrolysis of both nucleotides was completed. In contrast the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on

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