Detailed purine salvage metabolism in and outside the free malarial parasite

Purine utilization in the malarial parasite dependent on a “salvage” pathway was studied to determine the detailed mechanism of how purines were utilized and which precursor might be penetrating the membrane of the parasite.Erythrocyte-free malarial parasites (Plasmodium berghei) were incubated at 20 C with 2,8-³H-adenosine as a precursor for purine metabolism. Parasites and medium were separated using a unique system whereby the metabolites associated with the parasite and those contained in the medium can be identified after as little as 15 sec–10 min of incubation. It was shown that ³H-adenosine is rapidly deaminated to inosine and then deribosylated to hypoxanthine. The distribution of radioactivity indicated that these events occurred on the surface or outside of the parasite, while conversion of hypoxanthine to form IMP, and subsequently to ATP occurred most probably inside the parasite. The results indicated that hypoxanthine may be the immediate precursor entering the parasite membrane and is then converted to IMP eventually forming AMP, ADP, and ATP. ³H-IMP occurred in high concentration with a maximum occurring 2 min after incubation and gradually decreasing thereafter. The pool sizes of AMP and ADP appeared to be small and were quickly saturated. Formation of ³H-ATP continued to increase throughout the 10 min experimental period at which time > 80% of the added adenosine was converted to ATP. The large pool of IMP appeared to act as a “sink” to accomodate large amounts of purine intermediates available for later use and this could be a mechanism developed by the parasites to bypass the usual regulatory control of AMP.Phosphorylation and further utilization of ³H adenosine was completely eliminated in the presence of 5 × 10^?5M concentrations of adenosine, inosine, and hypoxanthine. Hypoxanthine, a normal-exit metabolite in mammalian purine metabolism, is apparently the building block of the nucleotides for the parasite indicating that hypoxanthine and/or its analogs may be able to antagonize and therefore have chemotherapeutic value in the treatment of malaria.Scanning-beam electron microscopy of the parasites showed that the free malarial parasites were round in shape measuring 1–2 μm (average 1.5 μm) in diameter and the outer surface appeared to be somewhat uneven.