Detection and characterization of DNA polymerase activity inToxoplasma gondii

Abstract

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites ofToxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase ofT. gondiiwas sensitive toN-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase ofT. gondii. Thus theT. gondiienzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.