A high-throughput screening for phosphatases using specific substrates.
A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.
Senn
Alejandro M
AM
Instituto Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina. asenn@leloir.org.ar
Wolosiuk
Ricardo A
RA
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000502:physiology; D029741:Brassica rapa / Q000201:enzymology*; D002736:Chloroplasts / Q000201:enzymology; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000201:enzymology*; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000502:physiology; D005636:Fructosephosphates / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000737:chemistry* / Q000502:physiology; D005958:Glucosephosphates / Q000378:metabolism; D016296:Mutagenesis; D019151:Peptide Library; D013379:Substrate Specificity
D005636:Fructosephosphates; D005958:Glucosephosphates; D019151:Peptide Library; C027618:fructose-6-phosphate; C031590:glucose-1-phosphate; D000469:Alkaline Phosphatase; D006597:Fructose-Bisphosphatase; D005952:Glucose-6-Phosphatase