Adenosine 3′,5′-monophosphate phosphodiesterase assay in tissue homogenates

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Abstract

  • 1.

    1. A fast, reliable and sensitive method for the assay of 3′,5′-AMP phosphodiesterase (adenosine 3′,5′-monophosphate phosphohydrolase, EC 3.1.4.c) is described, which is also applicable to homogenates and crude enzyme preparations containing enzymes catalyzing the breakdown of the reaction product 5′-AMP. The method has been worked out for rat pancreatic homogenate.

  • 2.

    2. The method is based on existing methods in which 3′,5′-[3H]AMP is used as substrate and in which the resulting 5′-AMP is dephosphorylated in a second step by means of added 5′-nucleotidase.

  • 3.

    3. It is shown that already during the first incubation step a mixture of radioactive products is formed: 5′-AMP, 5′-IMP, 3′,5′-IMP, adenosine, inosine, hypoxanthine and adenine. During the second incubation step 5′-AMP and 5′-IMP are converted into the corresponding nucleosides.

  • 4.

    4. The reaction mixture after the second incubation step is applied to a column of anionic exchange resin. All products of the phosphodiesterase reaction can be eluted with 0.1 M NaHCO3, while the unchanged 3′,5′-AMP and the product of the side reaction 3′,5′-IMP, are retained. After liquid scintillation counting of the eluate, the 3′,5′-AMP phosphodiesterase activity can be calculated.

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