title: A recycling assay for alkaline phosphatase applied to studies on its transport in E. coli K12.
abstract: A recycling assay for alkaline phosphatase, based on its ability to hydrolyse NADP to NAD+, is presented. The product NAD+ is recycled in a coupled assay consisting of NADH regeneration and reduction of a nitroblue tetrazolium salt. This assay is 10-12 times more sensitive than the conventional assay. We demonstrate the role of energy poisons in transport of this protein into the periplasm by combining the improved detection with phase separation of the periplasmic and cytoplasmic alkaline phosphatase pools.
authors.affiliation: Center for Cellular and Molecular Biology, Hyderabad, India.
authors.forename: N M
authors.initials: NM
authors.lastname: Rao
authors.forename: R
authors.initials: R
authors.lastname: Nagaraj
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009580:Nitroblue Tetrazolium; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods
chemical_list: D009243:NAD; D009580:Nitroblue Tetrazolium; D000469:Alkaline Phosphatase