Detection and characterization of DNA polymerase activity in Toxoplasma gondii.
A DNA polymerase activity has been detected and characterized in crude extracts from tachyzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6.4 S, corresponding to an approximate molecular weight of 150,000 assuming a globular shape. Like mammalian DNA polymerase alpha, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases alpha, delta and epsilon and also cytosine-beta-D-arabinofuranoside-5'-triphosphate which is an inhibitor of alpha polymerase. The activity was inhibited by 2',3'-dideoxythymidine-5'-triphosphate which is an inhibitor of mammalian DNA polymerase beta and gamma. 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 alpha did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.
Makioka
A
A
Department of Microbiology, School of Biological and Biomedical Sciences, University of Technology, Sydney, New South Wales, Australia.
Stavros
B
B
Ellis
J T
JT
Johnson
A M
AM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016590:Aphidicolin / Q000494:pharmacology; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D004259:DNA-Directed DNA Polymerase / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D054306:Dideoxynucleotides; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D019384:Nucleic Acid Synthesis Inhibitors; D011188:Potassium / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000494:pharmacology; D014122:Toxoplasma / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D001085:Arabinofuranosylcytosine Triphosphate; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; C017216:2',3'-dideoxythymidine triphosphate; D011188:Potassium