Rapid assay of cyclic AMP phosphodiesterase and 5'-nucleotidase by means of chromatography on cellulose-nitrate membrane strips.
A simple chromatographic procedure with the use of modified cellulose-nitrate membrane strips, 80 x 40 mm, has been worked out for the rapid isotopic assay of cyclic AMP (cAMP) phosphodiesterase (EC 3.1.4.17) and 5'-AMP nucleotidase (EC 3.1.3.5) in crude extracts of various tissues from animals and plants. The assay is based on enzymatic conversion of the product to adenine, a relatively inert compound which, in contrast to cAMP and 5'-AMP, is strongly adsorbed by the cellulose-nitrate membrane. Due to this property rapid separation of adenine from the unconverted substrate (cAMP or 5'-AMP) is possible. Commercial 5'-nucleotidase and easily obtainable crude extract of adenosine nucleosidase from barley leaves are used as coupling enzymes for the phosphodiesterase assay. The assay of phosphodiesterase in 0.5-2 microliter of blood (10(-5) to 4.10(-5) units) has been demonstrated on several examples.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood*; D000243:Adenosine Deaminase / Q000097:blood*; D002648:Child; D002845:Chromatography / Q000379:methods; D005767:Gastrointestinal Diseases / Q000097:blood / Q000151:congenital; D006801:Humans; D009700:Nucleoside Deaminases / Q000097:blood*
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase