Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of a soluble form.
The cyclic AMP phosphodiesterase (PDE) activity in Leishmania mexicana is mainly located (>95%) in the soluble fraction of the cell. The intact parasite, as well as plasma membranes, showed PDE activity, probably indicating that at least part of the activity in the particulate fraction resides on the parasite cell surface, with its catalytic domain facing the extracellular moiety. For the first time, a highly specific cAMP phosphodiesterase (PDE) was purified from the soluble fraction to apparent homogeneity after a single step 2239-fold purification using pseudo-affinity chromatography on Cibacron Blue 3GA agarose. The enzyme was identified as a 61-kDa protein on SDS-PAGE, with a K(m) of 277 microM at 30 degrees C (optimum temperature). The native enzyme protein showed an apparent molecular size of approximately 200000 estimated by molecular sieve chromatography on Sephacryl S-300. Further characterization of the PDE activity present in the soluble fraction shows that the enzyme requires Mg(2+) for maximal activity. Furthermore, no activity was detected when assayed at pHs below 6.0, but above this value it increased 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 rolipram, OPC-3911, cGMP, IBMX, zaprinast, theophylline, caffeine and Ca(2+)/calmodulin, 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 PDEs) similar to the those found in Candida albicans, Dictyostelium discoideum, Saccharomyces cerevisiae or Vibrio fischeri.
Rascón
A
A
Laboratory of Cell Signaling, Instituto de Biología Experimental, Universidad Central de Venezuela, Apartado 47.069, Caracas, Venezuela. rascona@hotmail.com
Viloria
M E
ME
De-Chiara
L
L
Dubra
M E
ME
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008970:Molecular Weight; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D015105:3',5'-Cyclic-AMP Phosphodiesterases