Abstract
A simple chromatographic procedure with the use of modified cellulose-nitrate membrane strips, 80 × 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 μl of blood (10 -5 to 4–10 -5 units) has been demonstrated on several examples.
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