Selective adsorption of 2'-O-anthraniloyl-AMP on DEAE-Sephadex: the basis of a direct, fluorescent assay for cyclic nucleotide phosphodiesterase.
The fluorescent, 2'-O-anthraniloyl derivative of AMP was selectively absorbed onto DEAE-Sephadex in the presence of zirconyl chloride in citrate buffer. Under these conditions 2'-O-anthraniloyl-cAMP was eluted from the column. The selective adsorption of the AMP derivative onto DEAE-Sephadex, in the presence of zirconyl chloride, was adapted to the direct discontinuous assay of cyclic nucleotide phosphodiesterase. In this assay the enzyme is incubated for 4 min with 2'-O-anthraniloyl-cAMP; after quenching of the reaction by boiling, zirconyl chloride is added and the product (2'-O-anthraniloyl-AMP) is separated from the substrate on a column (0.6 ml) of DEAE-Sephadex. 2'-O-Anthraniloyl-AMP is then eluted with NaCl (2 M) and quantitated spectrofluorometrically. Under the conditions employed, 2'-O-anthraniloyl-AMP concentrations as low as 0.1 nmol can be detected. In the present study, this assay has been used to estimate Km and Vmax values for 2'-O-anthraniloyl-cAMP hydrolysis catalyzed by highly purified, as well as crude, preparations of cyclic nucleotide phosphodiesterase from bovine brain.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D000327:Adsorption; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives; D003637:DEAE-Dextran; D006868:Hydrolysis; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D062367:ortho-Aminobenzoates
D062367:ortho-Aminobenzoates; D000249:Adenosine Monophosphate; C047403:2'-O-anthraniloyl cyclic AMP; D003637:DEAE-Dextran; C047402:2'-O-anthraniloyl adenosine monophosphate; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases