title: Direct determination of phosphatase activity from physiological substrates in cells.
abstract: A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France; State Key Laboratory of Supramolecular Structure and Materials, Jilin University Changchun, 130012, China.
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Ren
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France; Department of Biochemistry, Nencki Institute of Experimental Biology and Polish Academy of Sciences, 02-093 Warsaw, Poland.
authors.forename: Le Duy
authors.initials: le D
authors.lastname: Do
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Bechkoff
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: Saida
authors.initials: S
authors.lastname: Mebarek
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: Nermin
authors.initials: N
authors.lastname: Keloglu
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: Saandia
authors.initials: S
authors.lastname: Ahamada
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Meena
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: David
authors.initials: D
authors.lastname: Magne
authors.affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology and Polish Academy of Sciences, 02-093 Warsaw, Poland.
authors.forename: Slawomir
authors.initials: S
authors.lastname: Pikula
authors.affiliation: State Key Laboratory of Supramolecular Structure and Materials, Jilin University Changchun, 130012, China.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Université de Lyon, Villeurbanne, France; Université Lyon 1, Villeurbanne, France; INSA-Lyon, Villeurbanne, France; CPE Lyon, Villeurbanne, France; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Villeurbanne, France; CNRS UMR 5246, Villeurbanne, France.
authors.forename: René
authors.initials: R
authors.lastname: Buchet
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic; D045744:Cell Line, Tumor; D002642:Chick Embryo; D019902:Chondrocytes / Q000201:enzymology; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013055:Spectrophotometry, Infrared
chemical_list: D004791:Enzyme Inhibitors; D010744:Phosphoric Monoester Hydrolases