Partial purification and characterization of cyclic AMP phosphodiesterases from Funaria hygrometrica

Abstract

Cyclic AMP phosphodiesterase activity from chloronema cells of the moss Funaria hygrometrica has been purified 86-fold. This preparation contains more than a single phosphodiesterase activity since it hydrolyzes both 3', 5'-cAMP and 2', 3'-cAMP. It shows a high affinity (K_m = 8.7 ± 2.8 µ M) and a low affinity (K_m = 1.38 ± 0.37 mM) component for 3',5'-cAMP as substrate, but only one component (K_m = 0.58 ± 0.11 mM) for 2',3'-cAMP as substrate. The low K_m component is competitively inhibited by 2',3'-cAMP (K_i = 0.63 ± 0.04 mM). It has a molecular weight of 85,000, its activity is Zn²⁺ dependent, and is optimal at pH 5.5-6.5. Two peaks of enzyme activity are obtained upon further purification of the low K_m component of cAMP phosphodiesterase by affinity chromatography. Peak I activity, eluted by buffer, shows an acidic pH optimum, is insensitive to methylxanthines and imidazole, and copurifies with nucleotidases. Peak II activity binds to the affinity column and is eluted by 100 µ^M cAMP. This activity is free from nucleotidases, produces only 5'-AMP from cAMP, is maximally active at pH 7.5-8.0, is inhibited by methylxanthines, and is stimulated by imidazole. Thus peak II activity has properties similar to the cAMP phosphodiesterases present in animal tissues, while peak I resembles the phosphodiesterases from higher plants. The present work is the first clear evidence for the existence of both plant-like and animal-like phosphodiesterases in a plant development system.