1982. High-performance liquid chromatographic identification and quantitation of cyclic adenosine 3':5'-monophosphate in higher {Phaseotus vulgaris L. ) and lower (Chlorella sp.) plants. -Physiol. Plant. 55: 93-97.Cyclic adenosine 3':5'-monophosphate (cAMP) was extracted from Phaseolus vulgaris L. ev. Limburg seedlings and Chlorella sp. The cAMP was purified by charcoal adsorption, polyvinylpyrrolidone (PVP) and Dowex 50 W x 8 column chromatography and by preparative high-performanee liquid chromatography (HPLC). Quantitation was achieved by combining phosphodiesterase (PDE) treatment with analytical HPLC (reversed phase ion-pair partition chromatography) and cAMP-dependent protein kinase activation. This provided a verj' specific, accurate and sensitive assay i^or cAMP determinations. The eAMP content found in Chlorella (70-90 pmol/g dry wt) was comparable with previous reports using other quantitation methods, whereas the endogenous concentration found in bean seedlings (92 pmol/g dry wt) was considerably lower than previously reported data.Additional key words -cAMP, cAMP-dependent protein kinase, HPLC, phosphodiesterase.

Cyclic nucleotide phosphodiesterase (3′,5′‐cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH4)2SO4 precipitation, DEAE‐cellulose chromatography, chromatography on 3′,5′‐cAMP‐agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH4)2SO4 pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca2+, Mg2+ and Mn2+, whereas NaF, PPi and Fe3+ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An MI of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3′,5′‐cAMP‐agarose a phosphodiesterase was resolved that produced 5′‐AMP as sole reaction product.