Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors.
The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.
D000818:Animals; D000936:Antigen-Antibody Complex; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D008288:Malaria / Q000201:enzymology*; D010430:Pentosyltransferases / Q000235:genetics*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase