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Bone Abstracts (2014) 3 PP141 | DOI: 10.1530/boneabs.3.PP141


1Université de Lyon1, CPE Lyon, ICBMS CNRS UMR 5246, Villeurbanne, France; 2State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, China; 3Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.

Up to now, most of standard methods for measuring inorganic phosphate (Pi) to determine phosphatase activity are based on coupled enzyme assays, colorimetric methods or conductance measurements. However, none of these methods can measure Pi, substrate and protein concentrations simultaneously, allowing direct kinetic determinations of phosphatase activity in cells in a single assay. Among the enzymes having a pyrophosphatase activity and releasing Pi, is tissue non-specific alkaline phosphatase (TNAP) expressed in numerous tissues with high levels in bones, liver and neurons. TNAP activity is absolutely required for human life since severe functional deficiency is perinatally lethal. Chondrocytes, osteoblasts as well as smooth muscle cells during vascular calcification are characterized by high TNAP activity. Here we developed a continuous infrared (IR) assay to determine phosphatase activity in Saos-2 cells and in primary osteoblasts from exogenous substrates such as PPi, AMP, ADP, ATP, UTP, β-glycerophosphate or α-D-glucose 1-phosphate and para-nitrophenylphosphate (pNPP) Quantitative determinations were obtained for PPi, AMP, β-glycerophosphate, α-D-glucose 1-phosphate pNPP using both bands of Pi at 990 and 1070–1080 per cm. The substrate bands were also used to determine enzyme specific activity, as in the case of AMP (977 per cm), pNPP (980 per cm) and PPi (1107 per cm). At the concentrations of 5 mM, levamisole almost completely inhibited the PPi hydrolysis by Saos-2 cells (IC50=1.16±0.03 mM), suggesting TNAP contributed to the pyrophosphatase activity. A pyrophosphatase activity in matrix vesicles extracted from growth plates and epihyseal cartilage of 17-day-chicken embryos amounted to 1132±370 nmol/min per mg which is 10 times higher than in Saos-2 cells (110 nmol/min per mg). Thus, the IR assay can be employed as a cost-effective label-free approach to determine the phosphatase activity from extracellular physiological substrates in whole cells. It may serve to screen the phosphatase inhibitors in TNAP-enriched cells.

Volume 3

European Calcified Tissue Society Congress 2014

Prague, Czech Republic
17 May 2014 - 20 May 2014

European Calcified Tissue Society 

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