Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.     

Induction of nucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes

Normal bovine erythrocytes have negligible ability to transport adenosine and related nucleosides across their cell membrane. However, infection with the intraerythrocytic parasite Babesia bovis was found to induce a nucleoside permeation site into the host cell membrane. Transport experiments over periods of up to 30 s determined that the transport rate of 1 microM adenosine into the infected cell was 1.72 +/- 1.2 pmol incorporated (microliter cell water)-1s-1, a rate three times higher than for normal human erythrocytes. Incorporation studies over 6 h with labelled adenosine indicated that the purine moiety was incorporated into parasite nucleic acids. The mammalian nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole inhibited the induced nucleoside transport mechanism in the Babesia-infected erythrocytes, though at higher concentrations than those required to inhibit normal human erythrocyte transport. An ID50 value for NBMPR of 0.36 microM was determined. Phloretin and 5'-p-fluorosulphonyl benzoyl adenosine-HCl (5FSBA) were also shown to be inhibitory, with ID50 values of 0.11 and 0.18 microM, respectively, whilst phlorizin and verapamil at 1 microM had no effect. Binding studies with [3H]NBMPR indicated that high-affinity NBMPR binding sites could not be detected in either normal or B. bovis infected bovine erythrocytes. The results indicate that the induced nucleoside permeation site(s) in B. bovis infected erythrocytes has characteristics different from either human erythrocytes or erythrocytes infected with the malarial parasites Plasmodium falciparum or Plasmodium yoelii.     

Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.     

Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes

Impaired lymphocyte function is a common feature of human diabetes. Nucleoside transport across the plasma membrane is an essential step during lymphocyte growth and activation. In our study, we evaluated the impact of diabetic conditions on nucleoside transport system in B lymphocytes. Examination of the nucleoside transporters expression level in B lymphocytes isolated from diabetic rats revealed significant changes in their mRNA levels. Experiments performed on B cells cultured in medium containing defined concentrations of glucose and insulin showed that the rENT1 mRNA level was sensitive to extracellular glucose concentration and was not affected by insulin. Increase of glucose concentration from 5 to 20 mM caused a decrease of rENT1 mRNA by 80% and was associated with decreased adenosine uptake by the B cells. The effect of glucose was blocked by PD98059, a MAPK kinase inhibitor. The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but not on glucose concentration. Exposure of lymphocytes to 10 nM insulin resulted in a 2-fold increase in rENT2 mRNA and a 50% decrease in the rCNT2 mRNA level. Alterations in mRNA levels of rENT2 and rCNT2 were associated with changes in adenosine transport. Insulin-induced changes in expression level of rENT2 were blocked by wortmannin, an inhibitor of phosphatidylinositide 3-kinase, whereas the effect of insulin on rCNT2 was inhibited by PD98059 and to a lesser extend by wortmannin. In summary, impaired nucleoside transport in diabetic B lymphocytes results from alterations in the expression of nucleoside transporters, which are independently and differentially regulated by glucose and insulin.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes

The permeability properties of the membrane of human erythrocytes infected with malaria parasites (Plasmodium falciparum) were studied by the method of osmotic hemolysis. At the trophozoite stage, the host membrane becomes permeable to substrates such as sorbitol and glucose. The new permeability pathway is insensitive to most inhibitors of the glucose carrier, but is highly susceptible to the membrane dipole modifier phloretin. It is blocked by disaccharides and oligosaccharides, both of which are impermeant to non-infected and infected cells. It has an enthalpy of activation of solute penetration of 10 +/- 1 kcal mol-1 (range of 5-37 degrees C). It appears that new permeability pathways with pore-like properties are induced in parasitized cells. The pore(s) admit(s) neutral and anionic substances of a discrete molecular volume, but exclude(s) cations. Apparently they play an essential role in parasite development.     

All nucleoside transporters in bovine chromaffin cells are nitrobenzylthioinosine sensitive

Nitrobenzylthioinosine is an effective inhibitor of adenosine transport in chromaffin cells. When adenosine transport was measured at a 0.15 microM adenosine concentration, in the presence of variable concentrations of nitrobenzylthioinosine, ranging from 10(-14) to 10(-6) M, a half-maximal inhibitory concentration value (IC50) of 1 +/- 0.3 nM was deduced. This compound has the capacity to inhibit the total adenosine transport at 10(-7) M concentration. Nitrobenzylthioinosine acts in a non-competitive manner in blocking adenosine transport, as deduced from a Dixon plot, with a constant inhibition value (K1) of 0.01 +/- 0.003 nM. The results suggest that all nucleoside transporters present in bovine chromaffin cells are sensitive to the nitrobenzylthioinosine inhibition.     

Identification, expression and characterisation of a Babesia bovis hexose transporter

Babesia are tick-transmitted haemoprotozoan parasites that infect cattle, with an estimated 500 million at risk worldwide. Here, two predicted hexose transporters (BboHT1 and 2) have been identified within the Babesia bovis genome. BboHT1, having 40% and 47% amino acid sequence similarity compared with the human (GLUT1) and Plasmodium falciparum (PfHT) hexose transporters, respectively, is the only one that could be characterised functionally after expression in Xenopus laevis oocytes. Radiotracer studies on BboHT1 showed that it is a saturable, Na(+)-independent, stereo-specific hexose transporter, with a K(m) value for glucose of 0.84+/-0.54mM (mean+/-SEM). Using d-glucose analogues, hydroxyl positions at O-4 and O-6 have been identified as important for ligand binding to BboHT1. d-Glucose transport was inhibited maximally by cytochalasin B (50muM). A long-chain O-3 hexose derivative (compound 3361) that selectively inhibits PfHT also inhibited relatively potently BboHT1, with an apparent K(i) value of 4.1+/-0.9muM (mean+/-SEM). Compound 3361 did not inhibit B. bovis proliferation in in vitro growth assays but inhibited invasion of glucose-depleted bovine erythrocytes. Taken together with results of inhibition studies with cytochalasin B and beta-glucogallin, these data provide new insights into glucose metabolism of erythrocytic-stage Babesia infections.     

Effects of hexoses and anions on the erythritol permeability of human red cells

1. The effect of hexoses and of the anions chloride, thiocyanate, and salicylate on the permeability of human red cells to [(14)C]erythritol has been studied.2. It was confirmed that erythritol competes with glucose, mannose, and galactose for the hexose transfer system of the red cell membrane. Approximately 25% of the erythritol influx was insensitive to the presence of hexoses or phloretin. Identical maximum degrees of inhibition were obtained with 0.3 M glucose and with phloretin (0.5 x 10(-3)M). In the absence of competing inhibitors the erythritol permeability, P, was 1.2 x 10(-7) cm/sec at 38 degrees C. At maximum inhibition P was 0.3 x 10(-7) cm/sec.3. Erythritol is able to penetrate the membrane by two pathways, only one of which is sensitive to hexoses. Both hexose-sensitive and hexose-insensitive erythritol influx are well described by first-order diffusion kinetics. The affinity of erythritol for the hexose transfer system is very low, and the half saturation constants of hexoses can be determined from their ability to retard erythritol permeation. The following values were found for the half saturation of the transport system with hexoses at 38 degrees C: glucose 6 mM, mannose 11 mM, and galactose 40 mM.4. Thiocyanate and salicylate reduce the hexose-sensitive fraction of erythritol influx, but the hexose-insensitive erythritol permeability is not affected when chloride is replaced by the foreign anions. This applies to the whole temperature range between 0 and 38 degrees C, where the ionic permeabilities of red cells have been shown to be profoundly changed by thiocyanate and salicylate.     

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.     

Uridine transport in human erythrocytes: data from normal subjects and from patients with renal failure

Erythrocyte uridine transport has been studied in eight normal individuals and eight patients on haemodialysis for chronic renal failure. The initial rate of zero-trans uridine influx at 37 degrees C has been measured as a function of extracellular uridine concentration using [14C]-labelled uridine. The results are consistent with Michaelis-Menten kinetics. In normal humans the mean Vmax for uridine influx was 32.8 +/- 6.4 mumol (1 cells)-1 s-1 (mean +/- S.D.) and the mean Km was 190 +/- 12.3 microM. The measurements made in renal failure patients were not significantly different (mean Vmax 30.1 +/- 7.1 mumol (1 cells)-1 s-1, mean Km, 185 +/- 13.2 microM). These results are discussed with reference to the reported data on uridine transport in human erythrocytes at temperatures between 4 and 35 degrees C; it is suggested that zero-trans uridine influx shows a decrease in temperature dependence above 25 degrees C. The Vmax for zero-trans uridine influx at 37 degrees C in normal erythrocytes represents a turnover number for the nucleoside transporter of 180 uridine molecules per second.     

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells

Glucose concentrations of normal human airway surface liquid are ~12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of d-glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5–10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.     

N-ethylmaleimide differentially affects adenosine and thymidine uptake by rat thymocytes

The involvement of membrane sulfhydryl groups in the uptake of adenosine and thymidine was examined in rat thymocytes pretreated with 6,6'-dithiodinicotinic acid (CPDS) and N-ethylmaleimide (NEM). CPDS, which is known to react uniquely with external membrane sulfhydryls, under short incubation conditions, did not significantly affect the uptake of adenosine and thymidine. Formation of cAMP in nonstimulated and adenosine-stimulated cells was also unaltered by CPDS. However, inhibition of adenosine uptake by competitive inhibitor, dipyridamole, was significantly stronger when the cells were pretreated with CPDS. Preincubation of cells with NEM showed differential sensitivity of adenosine and thymidine uptake, depending on concentration of this sulfhydryl alkylating agent. The results suggest the involvement of NEM-accessible sulfhydryls in membrane transport of adenosine and thymidine. Dual effect of NEM on nucleoside transport may be related to the complexity of nucleoside carrier(s) or to the existence of different nucleoside carriers within thymocyte membranes. On the other hand, the easily accessible, external membrane -SH groups which can be blocked with CPDS, are not essential in thymocyte nucleoside transport but they appear to be situated at a site which interacts with the membrane transport system of nucleoside.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

Substrate depletion upregulates uptake of myo-inositol, glucose and adenosine in Leishmania

Leishmania flagellates undergo a digenetic life cycle in the gut of the sandfly insect vector and in macrophage phagolysosomes of the mammalian host. This involves vast changes of the environment to which the parasite has to adapt, including temperature, pH and concentration of nutrients between different types of meals of the insect vector or within the enclosed intracellular environment of the phagolysosome. The regulation of transporters for important organic substrates in Leishmania donovani, Leishmania mexicana and Leishmania enriettii has been investigated. A pronounced upregulation of inositol (25-fold), adenosine (11-fold) or glucose (5-fold) uptake activities was found when cells were depleted of the respective substrates during culture. Inositol-depleted cells showed a half-maximal uptake rate at nanomolar inositol concentration. Depletion of inositol only affected inositol uptake but did not affect uptake of glucose analog or proline in control experiments, indicating the specificity of the mechanism(s) underlying transport regulation. Adenosine-depleted cells showed an approximately 10-fold increase in both adenosine and uridine uptake, both mediated by the L. donovani nucleoside transporter 1 (LdNT1), but no change in guanosine uptake, which is mediated by the L. donovani nucleoside transporter 2 (LdNT2). These results suggest that extracellular adenosine concentration specifically regulates LdNT1 transport activity and does not affect LdNT2. The data imply that upregulation of transport activities by substrate depletion is a general phenomenon in protozoan flagellates, which is in remarkable contrast to bacteria where upregulation typically follows an increase of extracellular organic substrate. Hence, the parasites can maximize the uptake of important nutrients from the host even under limiting conditions, whereas bacteria often have dormant stages (spores) to overcome unfavorable environmental conditions or are heterotrophic for organic substrates.     

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.     

The accumulation and metabolism of a fluorescent ceramide derivative in Plasmodium falciparum-infected erythrocytes.

We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37 degrees C for 30 min, virtually all of the ceramide was converted to N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapble of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.     

Host urokinase-type plasminogen activator participates in the release of malaria merozoites from infected erythrocytes

Malaria infection of red blood cells is associated with plasminogen activation. Surface immunofluorescence and immunoprecipitation experiments, using specific polyclonal and monoclonal antibodies raised against human urokinase, demonstrate that this activity is due to the binding of host urokinase-type plasminogen activator to the surface of erythrocytes infected by mature forms of Plasmodium falciparum malaria parasites. Depletion of urokinase from the culture medium leads to the inhibition of merozoite release and the accumulation of segmenter-infected erythrocytes; this inhibition is reversed by the addition of human single-chain or two-chain urokinase. These findings are consistent with host urokinase being involved in the process of merozoite release from the red blood cell.     

Interferon enhanced human natural killer and antibody-dependent cell-mediated cytotoxic activity

Preincubation of normal human lymphocytes with human interferon for 18-24 h at 37 degrees C resulted in an increase of the activity of both natural killer (NK) cells and antibody-mediated cytotoxic cells (ADCC). The human myeloid line, K-562, which is highly susceptible to NK cells, was employed. ADCC was assessed with antibody-coated chick erythrocytes as targets. NK cells and ADCC were detected in a 4-hour 51Cr release assay. The magnitude of the enhancement was proportionate to the amount of interferon used in preincubation of the effector cells. Preincubation of tumor-target cells with interferon does not increase their susceptibility or resistance to lysis. The major effect of interferon on the cellular metabolism of the tumor-target cell is inhibition of DNA synthesis, but no direct cytotoxic effect was detected. Our findings may be important in understanding the mode of action of interferon in increasing host resistance to a variety of pathogens and tumors. This may be accomplished by inhibiting the growth of the tumor while simultaneously enhancing the natural killing mechanism for immunosurveillance.     

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.