Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of a soluble form

The cyclic AMP phosphodiesterase (PDE) activity in Leishmania mexicana is m ainly located (> 95%) in the soluble fraction of the cell. The intact paras ite, as well as plasma membranes, showed PDE activity, probably indicating that at least part of the activity in the particulate fraction resides on t he parasite cell surface, with its catalytic domain facing the extracellula r moiety. For the first time, a highly specific cAMP phosphodiesterase (PDE ) was purified from the soluble fraction to apparent homogeneity after a si ngle step 2239-fold purification using pseudo-affinity chromatography on Ci bacron Blue 3GA agarose. The enzyme was identified as a 61-kDa protein on S DS-PAGE, with a K-m of 277 mu M at 30 degrees C (optimum temperature). The native enzyme protein showed an apparent molecular size of approximate to 2 00 000 estimated by molecular sieve chromatography on Sephacryl S-300. Furt her characterization of the PDE activity present in the soluble fraction sh ows that the enzyme requires Mg2+ for maximal activity. Furthermore, no act ivity was detected when assayed at pHs below 6.0, but above this value it i ncreased dramatically, reaching the optimum at pH 7.2. On the basis of the K-m and PDE activity in presence of specific drugs or modulators such as ro lipram, OPC-3911, cGMP, IBMX, zaprinast, theophylline, caffeine and Ca2+/ca lmodulin, this enzyme does not seem to conform to any of the ten previously described Class I PDE families but to the PDE class II (or non-mammalian P DEs) similar to the those found in Candida albicans, Dictyostelium discoide um, Saccharomyces cerevisiae or Vibrio fischeri. (C) 2000 Published by Else vier Science B.V. All rights reserved.