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Assay of phosphodiesterase with radioactively labeled cyclic 3′,5′-AMP as substrate

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Summary

A highly sensitive method for the determination of cyclic 3′,5′-nucleotide phosphodiesterase (PDE) is described.

  1. 1.

    It is based on the use of radioactively labeled cyclic 3′,5′-AMP as substrate and the quantitative removal of the labeled product (3H-,14C-,32P-5′-AMP or32Pi).

  2. 2.

    3H- or14C-labeled 3′,5′-AMP can be used without further additions if the labeled product remains unchanged, e.g. withpurified enzyme preparations.

  3. 3.

    Whencrude PDE-preparations, which contain varying amounts of 5′-nucleotidase or phosphatase, are used either the labeled product of the PDE-reaction may be protected from further breakdown by the addition of high concentrations of unlabeled 5′-AMP, or the reaction may be carried out with32P-labeled substrate. The latter procedure is absolutely specific for measurements of PDE activity, since32Pi, formed by a subsequent step, is also quantitatively removed by precipitation.

    The effect ofaddition of 5′- AMP to the reaction mixture and the interference of 5′-nucleotidase activity was determined in various tissues.

  4. 4.

    Examples are given for certain characteristics of crude tissue PDE, for inhibition by drugsin vitro, and for the influence of pH and Mg2+ on the inhibition of PDE. Ki-values and the type of inhibition by drugs are described for crude as well as for purified PDE, and the differences are discussed.

  5. 5.

    Estimations of PDE in homogenized samples obtained fromisolated organs (heart, coronary arteries) are described.

  6. 6.

    The use, advantages and limitations of the method are discussed as well as the possible significance of effects of drugs on this key enzyme for their pharmacodynamic effects.

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Authors and Affiliations

  1. Pharmakologisches Institut der Universität Graz, Österreich

    G. Pöch

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The valuable technical help of Mrs. Vera Bauer is gratefully aknowledged. I am indebted to Prof. W. R. Kukovetz and to Prof. F. Hobbiger for helpful suggestions and criticism, and to Dr. R. H. Symons for a sample of32P-cyclic 3′,5′-AMP. I want to thank Dr. G. Michal for a gift of purified PDE and cyclic 3′,5′-AMP, Dr. K. G. Oldham for a research sample of14C-adenosine, and the various pharmaceutical companies for drug samples.

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Pöch, G. Assay of phosphodiesterase with radioactively labeled cyclic 3′,5′-AMP as substrate. Naunyn-Schmiedebergs Arch. Pharmak. 268, 272–299 (1971). https://doi.org/10.1007/BF00997262

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  • DOI: https://doi.org/10.1007/BF00997262

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