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Plasmodium falciparum: A Microassay for the Malarial Carbamoyl Phosphate Synthetase
EXPERIMENTAL PARASITOLOGY ARTICLE NO.
88, 243–245 (1998)
PR974240
RESEARCH BRIEF
Plasmodium falciparum: A Microassay for the Malarial Carbamoyl Phosphate Synthetase
Maria Vega C. Flores and Thomas S. Stewart School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, New South Wales 2052, Australia
Flores, M. V. C., and Stewart, T. S. 1998. Plasmodium falciparum: A microassay for the malarial carbamoyl phosphate synthetase. Experimental Parasitology 88, 243–245. q 1998 Academic Press Index Descriptors and Abbreviations: malaria; Plasmodium falciparum; CPS, carbamoyl phosphate synthetase; microassay; pfCPSII, P. falciparum carbamoyl phosphate synthetase II; PBS, phosphate buffered saline; NP-40, Nonidet P-40; PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide.
The mechanisms by which the human malarial parasite, Plasmodium falciparum, procures its nucleic acids for replication are potential loci for chemotherapy. Due to the inability to salvage pyrimidines from its environment, the parasite is solely dependent on de novo synthesis. The presence of the enzymes involved in the synthesis of uridine monophosphate in P. falciparum has been established (Reyes et al. 1982; Gero et al. 1984) but none have been purified to homogeneity due to major difficulties in obtaining large amounts of starting material. We have characterized the gene encoding the first enzyme of the pyrimidine pathway, carbamoyl phosphate synthetase (CPS) II (Flores et al. 1994). It resembles the conserved regions of other known CPSs except for the presence of two unique insert regions, a feature found in a number of P. falciparum genes including two that encode pyrimidine enzymes. The salient differences between the malarial CPS and the host counterpart represent putative targets for anti-malarials. This issue is being addressed by conventional drug design of protein antagonists and via nucleic acid therapy. Screening of test compounds for the specific inhibition of CPS activity requires a suitable assay that is easy, reproducible, and adapted to small culture volumes to reduce costs of drugs or synthetic oligonucleotides. CPS is known to be a very unstable
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
enzyme and the reported enzyme activity in P. falciparum by Gero et al. (1984) determined from 2 3 108 parasite cells was too low to be scaled down and detected in the drug sensitivity assays in 96-well microtiter plates with only 106 parasite cells per sample. The results reported by Gero et al. (1984) were obtained by an assay based on the procedure described by Tatibana and Shigesada (1972), where the conversion of Na[14C]HCO3 to [14C]carbamoyl phosphate and ultimately to [14C]citrulline in the presence of exogenous ornithine transcarbamoylase was measured. A specific activity of 0.04 nmol min21 mg protein21 (units) for nonsynchronous cultures or a maximum of 0.1 units for synchronous trophozoites was detected from crude protein extracts from large-scale parasite cultures. We report here a significant improvement on the previous results and an application to a microCPS assay. Experiments were first performed to optimize the assay conditions described by Gero et al. (1984) in a macroassay and then tested in smaller volumes of parasite culture with only 106 cells. FCQ-27 (Papua New Guinea) and K1 (Thailand) malaria strains were cultured as described (Trager and Jensen 1976) in 2% hematocrit. The protein extraction procedure was essentially carried out as described by Gero et al. (1984) but with two modifications: the use of detergent lysis (Grall et al. 1992) rather than the freeze/thaw method to liberate parasite cells from the erythrocyte host and extraction at room temperature prior to the addition of the cold dimethyl sulfoxide (DMSO)–glycerol solution. Synchronous ring-stage parasites at .5% parasitaemia (Lambros and Vanderberg 1979) were grown to mature trophozoites and harvested to obtain protein extracts. For macroassays, a 100-ml parasite culture was harvested by centrifugation at 3000g for 10 min. The pellet was lysed in 20 ml 0.02% saponin (prepared in phosphate-buffered saline (PBS)) for 10 min at room temperature. After centrifugation, the pellet was washed once with 0.02% (w/v) saponin and three times with PBS. Parasite lysate was prepared by resuspending the resulting pellet with 700 ml PBS containing 1.0% NP-40 and 2 mM phenylmethylsulfonyl fluoride (PMSF). The lysate
243
244 was transferred to a 1.5-ml microfuge tube and left for 5 min at room temperature. A cold DMSO–glycerol solution (300 ml; 75% (v/v) DMSO and 25% (w/v) glycerol in PBS) was mixed and the lysate kept on ice for 10 min. The preparation was clarified by microcentrifugation at 14,000 rpm for 10 min and the supernatant (1.0 ml) transferred to another microfuge tube. Storage of the crude lysates in 15% DMSO/ 5% glycerol for 2 weeks at 2208C did not alter pfCPSII activity. Protein concentrations were estimated by the Lowry method using Biorad DC protein assay kit. For microassays, 500 ml malaria cultures were harvested from 2 microtiter wells (96-well plates) and spun in a microfuge at low speed for 20 s. The pellet was lysed with 100 ml of PBS containing 1% NP40, 2 mM PMSF, 15% (v/v) DMSO, 5% (w/v) glycerol, and 2 mg/ml bovine serum albumin, incubated at room temperature for 10 min, and then kept on ice or at 2208C. No intermediate saponin lysis was carried out. Crude lysates from equal volumes of red blood cells were processed in the same way for control experiments. The Escherichia coli extracts used as controls were prepared from a 100-ml overnight culture. The cell pellet was resuspended in 5 ml PBS and lysed by sonication (2.5 output at 30% cycle for 2 min; Branson Sonifier 250). The clarified lysate was made up to 10% (w/v) glycerol. CPS activity was assessed by measuring the conversion of NaH14CO3 to [14C]carbamoyl phosphate in the presence of ATP, MgCl2, and L-glutamine and subsequently to [14C]citrulline by exogenous ornithine transcarbamoylase (Tatibana and Shigesada 1972; Shoaf and Jones 1973; Christopherson et al. 1981). The assay mixture contained 25 mM Hepes-KOH, pH 7.0; 20 mM ATP; 25 mM MgCl2 ; 3.3 mM Lglutamine; 0.5 mM L-ornithine; 1.0 mM dithiothreitol; ornithine transcarbamoylase (1 U); pyruvate kinase (10 ng), 7.5 mM phosphoenolpyruvate; and 100 ml crude enzyme. The reaction was performed in capped 1.5-ml microfuge tubes and started by adding 10 ml of 150 mM NaHCO3/NaH14CO3 (1.0 mCi/mmol; 1.5 3 106 dpm/mmol) to give a final concentration of 5 mM NaHCO3 in a total volume of 200 ml. The resulting concentrations of DMSO and glycerol of 7.5% (v/v) and 2.5% (w/v), respectively, were noted to maximize activity of mammalian CPSII (Tatibana and Shigesada 1972). Reaction mixtures were incubated at 378C for 15 min (macroassay) or 5 min (microassay) and terminated by transferring the total reaction volume to scintillation vials containing 100 ml of 5 M formic acid. The linearity of the reactions was established over these time periods from the two assays. A small piece of dry ice was added and the solution allowed to stand in a fume hood for 1 h to remove excess 14CO2. Distilled water (500 ml) and scintillant (5 ml; 2 parts toluene: 1 part Triton X-100, 0.5% 2,5diphenyloxazole, and 0.01% POPOP (1,4-bis[2-(5-phenyloxazoyl)]benzene; phenyl-oxazolylphenyl-oxazolyl-phenyl)) were added to the samples, which were then shaken vigorously and kept overnight in the dark before counting in a Tri-Carb 1900TR liquid scintillation spectrometer (Packard). The pfCPSII activity of 2.55 6 0.78 units (macroassay) and 3.61 6 0.01 units (microassay) determined from synchronous trophozoites from this study was more than 25-fold higher than that previously reported (Table I). The values are 60-fold higher than that detected from nonsynchronous parasites using the protocol of Gero et al. (1984). The substantive difference appears to be mainly due to the method of preparation of the crude extract used, particularly the detergent solubilization (NP-40) to obtain parasite lysate at room temperature. In confirmation, we also detected very low enzyme activity (0.085 6 0.04 units) using several rounds of freezing and thawing to release parasite protein. The present protocol appears to have improved the stability of pfCPSII. The mammalian enzyme has been observed to
FLORES AND STEWART
TABLE I CPSII Activities from P. faciparum Obtained Using Various Protocols as Compared to Other Organisms P. falciparum CPSII crude protein extraction procedures Detergent lysis/Macroassaya (synchronous parasites) Detergent lysis/Microassaya (synchronous parasites) Freeze/thaw procedure/Macroassay (nonsynchronous parasites; Gero et al. 1984) Freeze/thaw procedure/Macroassay (synchronous parasites; Gero et al. 1984) Freeze/thaw procedure/Macroassaya (synchronous parasites) CPSII activity from other sources Human red blood cellsa Escherichia colia Plasmodium berghei (Hill et al. 1981) Leishmania major (Hill et al. 1981) Toxoplasma gondii (Hill et al. 1981) a
Specific activity (No. of experiments) (nmol min21 mg protein21) 2.55 6 0.78 (6) 3.61 6 0.01 (5) 0.04 6 0.004 (2) 0.1 0.085 6 0.04 (3) 0.0002 6 0.00008 (4) 0.76 6 0.04 (3) 0.1 4.8 1.0
From this study.
almost entirely lose activity below 108C (Tatibana and Shigesada 1972). However, subsequent storage of the parasite enzyme at 15% DMSO/ 5% glycerol maintained enzyme activity below 08C for at least 2 weeks. Addition of pyruvate kinase and phosphoenolpyruvate as an ATP regenerating system improved the reproducibility of the results detected from extracts prepared by the freeze/thaw method, but did not increase the activity. The background activity from erythrocytes, as observed by others, was negligible. The pfCPSII activity reported here is more consistent with estimates from other protozoans (Hill et al. 1981). The principal objective of this study was to adapt the pfCPSII assay to volumes of malaria culture as small as 500 ml in order to test potential antimetabolites in microtiter plates. The earlier reported pfCPSII value was estimated from 108 parasites while this microassay can detect activity from only 106 parasites. Levels of expression of CPS from ring-stage parasites through to schizonts were followed using the microassay and correlated well with the findings of Gero et al. (1984). Monitoring the specific effects of antagonists on the CPS protein or gene can be easily carried out in parasite cultures from microtiter plates. This allows handling of more samples and simultaneous assays at one time as well as more cost-effective antimalarial tests. The procedure has been successfully used to assess the effect of ribozymes directed against the pfCPSII gene on enzyme production (Flores et al. 1997). (We thank Prof. W. J. O’Sullivan for his continued interest in the work and for revising the manuscript and Simone Berger for her technical assistance. This study was supported by Johnson & Johnson Research Pty Ltd.)
Plasmodium falciparum: A MICROASSAY FOR THE MALARIAL CARBAMOYL PHOSPHATE SYNTHETASE
REFERENCES
245
D. 1981. The enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Molecular and Biochemical Parasitology 2, 123–134.
Christopherson, R. I., Yu, M-L, and Jones, M. E. 1981. An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis. Analytical Biochemistry 111, 240–249.
Lambros, C., and Vanderberg, J. P. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology 65, 418–420.
Flores, M. V. C., Atkins, D., Wade, D., O’Sullivan, W. J., and Stewart, T. S. 1997. Inhibition of the proliferation of Plasmodium falciparum in vitro by ribozymes. Journal of Biological Chemistry, 272, 16940– 16945.
Reyes, P., Rathod, P. K., Sanchez, D. J., Mrema, J. E. K., Rieckmann, K. H., and Heidrich, H. G. 1982. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 5, 275–290.
Flores, M. V. C., O’Sullivan, W. J., and Stewart, T. S. 1994. Characterisation of the carbamoyl phosphate synthetase gene from Plasmodium falciparum. Molecular and Biochemical Parasitology 68, 315–318.
Shoaf, W. T., and Jones, M. E. 1973. Uridylic acid synthesis in Ehrlich Ascites carcinoma. Properties, subcellular distribution and nature of enzyme complexes of the six biosynthetic enzymes. Biochemistry 12, 4039–4051.
Gero, A. M., Brown, G. V., and O’Sullivan, W. J. 1984. Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. Journal of Parasitology 70, 536–541. ¨ Grall, M., Srivastava, I. K., Schmidt, M., Garcia, A., Mauel, J., and Perrin, L. H. 1992. Plasmodium falciparum: Identification and purification of the phosphoglycerate kinase of the malaria parasite. Experimental Parasitology 75, 10–18. Hill, B., Kilsby, J., Rogerson, G. W., McIntosh, R. T., and Ginger, C.
Tatibana, M., and Shigesada, K. 1972. Control of pyrimidine biosynthesis in mammalian tissues. IV. Requirements of a quantitative assay of glutamine-dependent CPS and effect of magnesium ion as an essential activator. Journal of Biochemistry (Tokyo) 72, 537–547. Trager, W., and Jensen, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 673–675. Received 24 March 1997; accepted with revision 17 September 1997
88, 243–245 (1998)
PR974240
RESEARCH BRIEF
Plasmodium falciparum: A Microassay for the Malarial Carbamoyl Phosphate Synthetase
Maria Vega C. Flores and Thomas S. Stewart School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, New South Wales 2052, Australia
Flores, M. V. C., and Stewart, T. S. 1998. Plasmodium falciparum: A microassay for the malarial carbamoyl phosphate synthetase. Experimental Parasitology 88, 243–245. q 1998 Academic Press Index Descriptors and Abbreviations: malaria; Plasmodium falciparum; CPS, carbamoyl phosphate synthetase; microassay; pfCPSII, P. falciparum carbamoyl phosphate synthetase II; PBS, phosphate buffered saline; NP-40, Nonidet P-40; PMSF, phenylmethylsulfonyl fluoride; DMSO, dimethyl sulfoxide.
The mechanisms by which the human malarial parasite, Plasmodium falciparum, procures its nucleic acids for replication are potential loci for chemotherapy. Due to the inability to salvage pyrimidines from its environment, the parasite is solely dependent on de novo synthesis. The presence of the enzymes involved in the synthesis of uridine monophosphate in P. falciparum has been established (Reyes et al. 1982; Gero et al. 1984) but none have been purified to homogeneity due to major difficulties in obtaining large amounts of starting material. We have characterized the gene encoding the first enzyme of the pyrimidine pathway, carbamoyl phosphate synthetase (CPS) II (Flores et al. 1994). It resembles the conserved regions of other known CPSs except for the presence of two unique insert regions, a feature found in a number of P. falciparum genes including two that encode pyrimidine enzymes. The salient differences between the malarial CPS and the host counterpart represent putative targets for anti-malarials. This issue is being addressed by conventional drug design of protein antagonists and via nucleic acid therapy. Screening of test compounds for the specific inhibition of CPS activity requires a suitable assay that is easy, reproducible, and adapted to small culture volumes to reduce costs of drugs or synthetic oligonucleotides. CPS is known to be a very unstable
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
enzyme and the reported enzyme activity in P. falciparum by Gero et al. (1984) determined from 2 3 108 parasite cells was too low to be scaled down and detected in the drug sensitivity assays in 96-well microtiter plates with only 106 parasite cells per sample. The results reported by Gero et al. (1984) were obtained by an assay based on the procedure described by Tatibana and Shigesada (1972), where the conversion of Na[14C]HCO3 to [14C]carbamoyl phosphate and ultimately to [14C]citrulline in the presence of exogenous ornithine transcarbamoylase was measured. A specific activity of 0.04 nmol min21 mg protein21 (units) for nonsynchronous cultures or a maximum of 0.1 units for synchronous trophozoites was detected from crude protein extracts from large-scale parasite cultures. We report here a significant improvement on the previous results and an application to a microCPS assay. Experiments were first performed to optimize the assay conditions described by Gero et al. (1984) in a macroassay and then tested in smaller volumes of parasite culture with only 106 cells. FCQ-27 (Papua New Guinea) and K1 (Thailand) malaria strains were cultured as described (Trager and Jensen 1976) in 2% hematocrit. The protein extraction procedure was essentially carried out as described by Gero et al. (1984) but with two modifications: the use of detergent lysis (Grall et al. 1992) rather than the freeze/thaw method to liberate parasite cells from the erythrocyte host and extraction at room temperature prior to the addition of the cold dimethyl sulfoxide (DMSO)–glycerol solution. Synchronous ring-stage parasites at .5% parasitaemia (Lambros and Vanderberg 1979) were grown to mature trophozoites and harvested to obtain protein extracts. For macroassays, a 100-ml parasite culture was harvested by centrifugation at 3000g for 10 min. The pellet was lysed in 20 ml 0.02% saponin (prepared in phosphate-buffered saline (PBS)) for 10 min at room temperature. After centrifugation, the pellet was washed once with 0.02% (w/v) saponin and three times with PBS. Parasite lysate was prepared by resuspending the resulting pellet with 700 ml PBS containing 1.0% NP-40 and 2 mM phenylmethylsulfonyl fluoride (PMSF). The lysate
243
244 was transferred to a 1.5-ml microfuge tube and left for 5 min at room temperature. A cold DMSO–glycerol solution (300 ml; 75% (v/v) DMSO and 25% (w/v) glycerol in PBS) was mixed and the lysate kept on ice for 10 min. The preparation was clarified by microcentrifugation at 14,000 rpm for 10 min and the supernatant (1.0 ml) transferred to another microfuge tube. Storage of the crude lysates in 15% DMSO/ 5% glycerol for 2 weeks at 2208C did not alter pfCPSII activity. Protein concentrations were estimated by the Lowry method using Biorad DC protein assay kit. For microassays, 500 ml malaria cultures were harvested from 2 microtiter wells (96-well plates) and spun in a microfuge at low speed for 20 s. The pellet was lysed with 100 ml of PBS containing 1% NP40, 2 mM PMSF, 15% (v/v) DMSO, 5% (w/v) glycerol, and 2 mg/ml bovine serum albumin, incubated at room temperature for 10 min, and then kept on ice or at 2208C. No intermediate saponin lysis was carried out. Crude lysates from equal volumes of red blood cells were processed in the same way for control experiments. The Escherichia coli extracts used as controls were prepared from a 100-ml overnight culture. The cell pellet was resuspended in 5 ml PBS and lysed by sonication (2.5 output at 30% cycle for 2 min; Branson Sonifier 250). The clarified lysate was made up to 10% (w/v) glycerol. CPS activity was assessed by measuring the conversion of NaH14CO3 to [14C]carbamoyl phosphate in the presence of ATP, MgCl2, and L-glutamine and subsequently to [14C]citrulline by exogenous ornithine transcarbamoylase (Tatibana and Shigesada 1972; Shoaf and Jones 1973; Christopherson et al. 1981). The assay mixture contained 25 mM Hepes-KOH, pH 7.0; 20 mM ATP; 25 mM MgCl2 ; 3.3 mM Lglutamine; 0.5 mM L-ornithine; 1.0 mM dithiothreitol; ornithine transcarbamoylase (1 U); pyruvate kinase (10 ng), 7.5 mM phosphoenolpyruvate; and 100 ml crude enzyme. The reaction was performed in capped 1.5-ml microfuge tubes and started by adding 10 ml of 150 mM NaHCO3/NaH14CO3 (1.0 mCi/mmol; 1.5 3 106 dpm/mmol) to give a final concentration of 5 mM NaHCO3 in a total volume of 200 ml. The resulting concentrations of DMSO and glycerol of 7.5% (v/v) and 2.5% (w/v), respectively, were noted to maximize activity of mammalian CPSII (Tatibana and Shigesada 1972). Reaction mixtures were incubated at 378C for 15 min (macroassay) or 5 min (microassay) and terminated by transferring the total reaction volume to scintillation vials containing 100 ml of 5 M formic acid. The linearity of the reactions was established over these time periods from the two assays. A small piece of dry ice was added and the solution allowed to stand in a fume hood for 1 h to remove excess 14CO2. Distilled water (500 ml) and scintillant (5 ml; 2 parts toluene: 1 part Triton X-100, 0.5% 2,5diphenyloxazole, and 0.01% POPOP (1,4-bis[2-(5-phenyloxazoyl)]benzene; phenyl-oxazolylphenyl-oxazolyl-phenyl)) were added to the samples, which were then shaken vigorously and kept overnight in the dark before counting in a Tri-Carb 1900TR liquid scintillation spectrometer (Packard). The pfCPSII activity of 2.55 6 0.78 units (macroassay) and 3.61 6 0.01 units (microassay) determined from synchronous trophozoites from this study was more than 25-fold higher than that previously reported (Table I). The values are 60-fold higher than that detected from nonsynchronous parasites using the protocol of Gero et al. (1984). The substantive difference appears to be mainly due to the method of preparation of the crude extract used, particularly the detergent solubilization (NP-40) to obtain parasite lysate at room temperature. In confirmation, we also detected very low enzyme activity (0.085 6 0.04 units) using several rounds of freezing and thawing to release parasite protein. The present protocol appears to have improved the stability of pfCPSII. The mammalian enzyme has been observed to
FLORES AND STEWART
TABLE I CPSII Activities from P. faciparum Obtained Using Various Protocols as Compared to Other Organisms P. falciparum CPSII crude protein extraction procedures Detergent lysis/Macroassaya (synchronous parasites) Detergent lysis/Microassaya (synchronous parasites) Freeze/thaw procedure/Macroassay (nonsynchronous parasites; Gero et al. 1984) Freeze/thaw procedure/Macroassay (synchronous parasites; Gero et al. 1984) Freeze/thaw procedure/Macroassaya (synchronous parasites) CPSII activity from other sources Human red blood cellsa Escherichia colia Plasmodium berghei (Hill et al. 1981) Leishmania major (Hill et al. 1981) Toxoplasma gondii (Hill et al. 1981) a
Specific activity (No. of experiments) (nmol min21 mg protein21) 2.55 6 0.78 (6) 3.61 6 0.01 (5) 0.04 6 0.004 (2) 0.1 0.085 6 0.04 (3) 0.0002 6 0.00008 (4) 0.76 6 0.04 (3) 0.1 4.8 1.0
From this study.
almost entirely lose activity below 108C (Tatibana and Shigesada 1972). However, subsequent storage of the parasite enzyme at 15% DMSO/ 5% glycerol maintained enzyme activity below 08C for at least 2 weeks. Addition of pyruvate kinase and phosphoenolpyruvate as an ATP regenerating system improved the reproducibility of the results detected from extracts prepared by the freeze/thaw method, but did not increase the activity. The background activity from erythrocytes, as observed by others, was negligible. The pfCPSII activity reported here is more consistent with estimates from other protozoans (Hill et al. 1981). The principal objective of this study was to adapt the pfCPSII assay to volumes of malaria culture as small as 500 ml in order to test potential antimetabolites in microtiter plates. The earlier reported pfCPSII value was estimated from 108 parasites while this microassay can detect activity from only 106 parasites. Levels of expression of CPS from ring-stage parasites through to schizonts were followed using the microassay and correlated well with the findings of Gero et al. (1984). Monitoring the specific effects of antagonists on the CPS protein or gene can be easily carried out in parasite cultures from microtiter plates. This allows handling of more samples and simultaneous assays at one time as well as more cost-effective antimalarial tests. The procedure has been successfully used to assess the effect of ribozymes directed against the pfCPSII gene on enzyme production (Flores et al. 1997). (We thank Prof. W. J. O’Sullivan for his continued interest in the work and for revising the manuscript and Simone Berger for her technical assistance. This study was supported by Johnson & Johnson Research Pty Ltd.)
Plasmodium falciparum: A MICROASSAY FOR THE MALARIAL CARBAMOYL PHOSPHATE SYNTHETASE
REFERENCES
245
D. 1981. The enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Molecular and Biochemical Parasitology 2, 123–134.
Christopherson, R. I., Yu, M-L, and Jones, M. E. 1981. An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis. Analytical Biochemistry 111, 240–249.
Lambros, C., and Vanderberg, J. P. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology 65, 418–420.
Flores, M. V. C., Atkins, D., Wade, D., O’Sullivan, W. J., and Stewart, T. S. 1997. Inhibition of the proliferation of Plasmodium falciparum in vitro by ribozymes. Journal of Biological Chemistry, 272, 16940– 16945.
Reyes, P., Rathod, P. K., Sanchez, D. J., Mrema, J. E. K., Rieckmann, K. H., and Heidrich, H. G. 1982. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 5, 275–290.
Flores, M. V. C., O’Sullivan, W. J., and Stewart, T. S. 1994. Characterisation of the carbamoyl phosphate synthetase gene from Plasmodium falciparum. Molecular and Biochemical Parasitology 68, 315–318.
Shoaf, W. T., and Jones, M. E. 1973. Uridylic acid synthesis in Ehrlich Ascites carcinoma. Properties, subcellular distribution and nature of enzyme complexes of the six biosynthetic enzymes. Biochemistry 12, 4039–4051.
Gero, A. M., Brown, G. V., and O’Sullivan, W. J. 1984. Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. Journal of Parasitology 70, 536–541. ¨ Grall, M., Srivastava, I. K., Schmidt, M., Garcia, A., Mauel, J., and Perrin, L. H. 1992. Plasmodium falciparum: Identification and purification of the phosphoglycerate kinase of the malaria parasite. Experimental Parasitology 75, 10–18. Hill, B., Kilsby, J., Rogerson, G. W., McIntosh, R. T., and Ginger, C.
Tatibana, M., and Shigesada, K. 1972. Control of pyrimidine biosynthesis in mammalian tissues. IV. Requirements of a quantitative assay of glutamine-dependent CPS and effect of magnesium ion as an essential activator. Journal of Biochemistry (Tokyo) 72, 537–547. Trager, W., and Jensen, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 673–675. Received 24 March 1997; accepted with revision 17 September 1997