Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes

MOLECULAR AND

ELSEVIER

Molecular and Biochemical Parasitology 63 (1994) 179-192

BIOCHEMICAL PARASITOLOGY

Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositide cycle in Plasmodium-infected erythrocytes Noureddine Elabbadi, Marie Laure Ancelin, Henri Joseph Vial* Interactions Membranaires, CNRS URA. 530, Universitd Montpellier H case 107, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France

Received 19 July 1993; accepted 6 October 1993

Abstract Plasmodium knowlesi-infected erythrocytes possess a membranous cytidine 5'-diphospho-l,2-diacyl-sn-glycerol: myoinositol 3-phosphatidyl transferase (PI synthase) (EC 2.7.8.11) activity of 10 + 1.7 nmol min -~ per 10 ~° infected cells. The activity was successfully solubilized with 40 mM n-octyl-fl-D-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal pH was 8 and the apparent Ks for Mn 2 + was 0.1 raM. Mg 2+ allowed two-fold higher PI synthase activity, with an optimum above 100 raM. Calcium alone was ineffective while at 2 mM it inhibited solubilized PI synthase activity in the presence of 100 mM Mg 2+. Enzymatic activity was fully dependent on CDP-diacylglycerol and inositol with apparent Kr~ of 0.16+0.1 mM and 1+0.5 mM respectively. Affinity chromatography clearly showed CDP-diacylglycerol-dependent interactions of PI synthase with CDPdiacylglycerol Sepharose. However, elution of enzymatic activity in an active form was unsuccessful while SDS-PAGE of the eluate showed one apparent band. Incubations of Plasmodium falciparum-infected erythrocytes with 32p or [3H]inositol revealed de novo biosynthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate which appeared to predominate in the second half of the asexual cellular cycle. Ionomycin, a calcium ionophore, induced Li +-sensitive production of radioactive inositol phosphates, with neo-synthesized inositol 1,4,5-trisphosphate accumulation being the highest. Key words." Plasmodium falciparum; Plasmodium knowlesi; Lipid; Phosphatidylinositol synthase; Polyphosphoinositide cycle

Corresponding author. Tel.: + 33 67 14 37 45; Fax: + 33 67 14 42 86.

I. Introduction

Abbreviations: DAG, diacylglycerol; PA, phosphatidic acid; PL, phospholipid; PC, PE, PS, PI, phosphatidylcholine, ethanolamine, -serine, -inositol; PPI, polyphosphoinositide; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; IP, IP2, IP3, inositol monophosphate, bisphosphate, -trisphosphate; OG, n-octyl-/~-D-glucopyranoside; CMC, critical micellar concentration; t.l.c., thin-layer chromatography.

One m a i n characteristic in the b l o o d stage o f Plasmodium is the considerable increase in phospholipids (PLs) for the biogenesis o f its new m e m branes. M a t u r e erythrocytes show only a very low turnover o f m e m b r a n e lipids a n d are essentially devoid o f any newly biosynthesized lipids [1,2]. N e w P L molecules are synthesized de n o v o by the p a r a -

Elsevier Science B.V. SSDI 0 1 6 6 - 6 8 5 1 ( 9 3 ) E 0 1 6 6 - 6

180

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179 192

sitic enzymatic machinery from polar heads and fatty acids taken up from the plasma, while a minor part could directly originate from the plasma [3]. Phosphatidylinositol (PI), which is present in trace amounts in normal mammalian mature erythrocytes (less than 3% of total PL), experiences the highest relative increase after P. knowlesi or P. falciparum infection [3]. PI is of considerable importance in the growth of eukaryotic cells and generally in animal cells [4]. PI is most interesting since it serves as a precursor in the synthesis of polyphosphoinositide (PPI), i.e., phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) [4,5]. The key role of PPI, consisting of intermediates that transmit signals for a variety of hormones, neurotransmitters and growth factors, is now well established [5,6]. The primary products of this biochemical response, mainly inositol 1,4,5-trisphosphate (IP3) and DAG, act as potent second messengers controlling a great variety of cellular events including metabolism, proliferation and differentiation [6]. We thus investigated PI synthase activity, the enzyme responsible for biosynthesis of PI from CDP-diacylglycerol (CDP-DAG) and myo-inositol [7]. We also demonstrated that P. knowlesi and P. falciparum were able to synthesize PIP2 via a de novo pathway, and this biosynthesis from inositol was mainly observed at the schizont stage. In addition, infected erythrocytes contain an active inositol signalling pathway which is activated by a calcium ionophore, which suggests the existence of phospholipase C activity in Plasmodium.

2. Materials and methods

Myo-[2-3H]inositol and [3H]inositol monophosphate ([3H]IP) were purchased from Amersham Corp. (Bucks., UK) and carrier-free, neutral 32p was obtained from CEA (Saclay, France). N-octyl-J~-D-glucopyranoside (OG), ionomycin, cyanogen bromide-activated Sepharose 4B were from Sigma (St. Louis, USA). CDP-DAG was obtained from Serdary Research Laboratory (Montlu~on, France). RPMI 1640 and modified RPMI 1640 without choline, inositol, serine and methion-

ine were GIBCO France).

products

(Cergy Pontoise,

2.1. Biological materials. Human blood and type AB ÷ human serum were from the local blood bank (Montpellier, France). The Nigerian strain of P. falciparum [8] was maintained by serial passage in AB + human erythrocytes suspended in complete medium (RPMI 1640 supplemented with 25 mM Hepes buffer, pH 7.4, and 10% AB + serum) by the petri dish candle-jar methods of Jensen and Trager [9]. In some experiments, parasites were twice synchronized by 5% sorbitol treatments [10]. P. knowlesi (Washington strain, variant 1)-infected erythrocytes were harvested from splenectomized Macaca fascicularis monkeys (SANOFI, Montpellier, France) as already described [11]. Infected blood cells were suspended in modified RPMI supplemented with 25 mM Hepes, pH 7.4 (medium I), then passed through a cellulose powder column (CF l l, Whatman) to remove white blood cells [12]. Separation of infected and uninfected red blood cells was performed by the Percoll-sorbitol fractionation procedure [13]. After enrichment, parasitemia was close to 100% and white blood cells were practically absent (less than 1%o), as monitored by microscopic examination of Giemsa stained smears. In some cases, experiments were conducted with P. knowlesi-infected erythrocytes without further enrichment. In some cases, infected cells were stored at -20~C before the homogenization step. 2.2. Incubation procedures Jor PPI synthesis assessment. Studies were carried out at 37°C using the candle-jar method [9]. Incubation media were either Hepes-buffered saline solution supplemented with glucose (medium II): 137 mM NaCI/ 4 mM KCI/ 1.5 mM MgCIz and CaCI2/ 30 mM Hepes, pH 7.4/ 10 mM glucose, or modified RPMI 1640 (free of inositol and methionine, adjusted to 20 #M choline, 40 #M serine) supplemented with 25 mM Hepes-buffer, pH 7.4, (medium III). Medium III contained Ca 2+ at 0.5 raM. Unless otherwise stated, ceils were centrifuged at 1000 x g for 10 min and washed with 0.9% NaC1 at the end of the experiment time.

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179 192

2.3. Extraction of lipids. Lipids were extracted from the red blood cells according to the method of Folch modified by Rock et al. [14]. Extraction of PPI in acidic conditions was adapted from Schacht [15], the erythrocyte pellet (up to 200 #1 of packed cell volume) received 50/d of 100 mM EDTA, 1.2 ml of cold methanol followed by 1.2 ml chloroform. 150 ~tg of PPI from bovine brain (Sigma P.6023) was added to the combined lower phases. The extracted lipids were then separated by t.l.c. (thin-layer chromatography). Development of silica gel precoated plates (No. 11845, Merck, Germany) in chloroform/methanol/acetic acid/water (65:43:1:3, v/v) permitted clear separation of lyso-phosphatidylcholine (lyso-PC), sphingomyelin, phosphatidylcholine (PC), phosphatidylserine (PS), PI, phosphatidylethanolamine (PE) provided the plates were reactivated in vacuo in the presence of P205 as dessicator after sample applications. PPI were separated using chloroform/methanol/4 N NHnOH (9:7:2, v/v). Phosphatidic acid (PA), cardiolipids and PE were separated using chloroform/methanol/acetic acid/water (160:26:16:0.6, v/v). The lipids were visualized with iodine vapour and, in the case of 32p incorporation, by autoradiography. Lipid spots were scraped directly into scintillation vials and radioactivity was measured with a Beckman 380l liquid scintillation spectrometer using a Scintillator 299 (Packard, France). 2.4. Determination of water-soluble inositol phosphates. [3H]inositol phosphates were separated and quantified by anion exchange chromatography [16] of the aqueous phase obtained after stopping incubations with 2 ml ice-cold 5% perchloric acid. The neutralized aqueous phases were applied to a column (0.6 cm diameter) containing 2 ml of a 50% aqueous slurry of Dowex AG 1 x 8 suspension (200400 mesh, formate form Bio Rad, France). The column was then washed with an additional 10 ml water. Glycerophosphorylinositol was eluted with I0 ml 60 mM sodium formate and 5 mM disodium tetraborate. During this wash, eluted radioactivity fell to the background level. IP, inositol-bisphosphate (IPa), and IP3 were then sequentially eluted with 10 ml 0.1 M formic acid containing 0.2 M, 0.4 M and 1 M

181

ammonium formate, respectively. Radioactivity was determined by scintillation counting. Control studies using labeled standard showed that the elution pattern yielded 95% recovery of [3H]IP. This method clearly separates IP from IP2, and IP3, but does not distinguish their various possible isomers.

2.5. Preparation of cytosolic and membrane fractions of parasitized erythrocytes. All steps were carried out at 0-4°C. P. knowlesi-infected erythrocytes (0.5 ml packed cell volume, 100% infected cells) were suspended in 3 ml 50 mM Tris-HC1 buffer, pH 8, containing 0.1 M sucrose, 5 mM EDTA, 10 mM 2-mercaptoethanol, and proteinase inhibitors (50 ktg m1-1 L-trans-epoxysuccinyMeucilamido(4-amino)butane, leupeptin, N-c~p-tosyl-L-lysine chloromethylketone, chymostatin and g- 1-tosylamide-2-phenyl-ethylchloromethylketone; 0.35 mg m1-1 phenylmethylsulfonylfluoride; 20 pg m l - l pepstatin A) (medium A). The cell suspension was sonicated in a probe-type sonicator (Sonimasse $20) for 1 min at 4°C. Unbroken cells were removed by centrifugation at 2000 g for 10 min. The crude homogenate (H0) was then centrifuged at 100000 x g for 30 min (Beckman, Ti 50 rotor) to obtain the supernatant ($1) and pellet fraction. This pellet fraction was rinsed twice with 50 mM Tris-HC1 buffer (pH 8) and is hereafter referred to as P1. 2.6. Solubilization of PI synthase with n-octyl-fl-Dglucopyranoside . PI synthase activity was extracted from the membrane fraction (P1) with 1 ml of 50 mM Tris-HC1 buffer, pH 8, containing 2 mM MnC12, 30 mM MgC12, 0.5 M KC1, 10 mM 2mercaptoethanol, 20% glycerol (w/v) and 40 mM OG (medium B). The molar ratio of OG to total PL from the membrane fraction was around 4:1. After 30 min at 0-4°C, the suspension (H1) was centrifuged at 100 000 x g for 30 min to obtain the supernatant ($2) and pellet (P2). The pellet was usually resuspended in 0.5 ml of medium B and is hereafter referred to as H2. 2.7. Enzyme assays. Unless otherwise stated, PI synthase activity was measured at 30°C for 30 min by following the incorporation of 3 mM myo-[2-3H]inositol (1.7 Ci tool -1) into the lipid

182

N. Elabbadi et al./ Molecular and Biochemical Parasitology 63 (1994) 179 192

fraction in a medium containing 50 mM Tris-HCl buffer, pH 8, 100 m M MgC12, 0.2 m M CDPD A G , 30 m M OG, and usually 20 /~1 of solubilized cell-free extract (which corresponded to 3 10 x 107 P. knowlesi-infected erythrocytes) in a total volume of 0.2 ml. The PL product of the reaction was extracted using chloroform and methanol as described previously [11] and identified by t.l.c.. Radioactive profiles of the applied sample were determined by counting 1-cm strips. The radioactive product comigrated with standard PI with an Rv of 0.34 in a solvent system containing chloroform/methanol/acetic acid/0.1 M sodium borate (75:45:6:2, v/v).

2.8. Preparation of mixed micelle substrate. An adequate amount of C D P - D A G was solubilized in a 20 m M aqueous dispersion of O G followed by sonication for 2 rain at 4°C. The ratio of O G to C D P - D A G was always maintained at 5:1. 10-/~1 aliquots of this solution were added to the enzymatic reactional mixture to achieve 0.2 m M C D P - D A G final concentration and O G concentration in the reaction mixture was further adjusted. In experiments carried out with Triton X100 instead of OG, the preparation of mixed micellar solutions of C D P - D A G and the molar ratios was similar. 2.9. Affinity chromatography on CDP-DAG-Sepharose resin. The NaIO4-oxidized derivative C D P - D A G was covalently attached to Sepharose 4B via an adipic acid dihydrazide spacer arm as described previously [17]. Generally, two volumes of OG-solubilized enzymatic extract were mixed with one volume of the affinity resin (in batches) equilibrated with medium B. Elution was carried out with two-fold the resin volume of medium B supplemented with 2 m M C D P - D A G . Samples were analyzed on 10% polyacrylamide gel electrophoresis [18]. Proteins were determined by the Coomassie blue dye-binding method of Bradford [19] with BSA as standard.

3. Results

3.1. PI synthase activity assessment.

Extensive in-

corporation of [3H]inositol into P1 occurred when whole P. knowlesi homogenate H0 was incubated under the standard conditions of the PI synthase assay described in the Methods. After 30 min incubation, organic phase analysis on silica gel t a x . showed that more than 95% of the recovered radioactivity was associated with authentic PI. For these reasons, the activity was routinely measured by directly monitoring the incorporation of radioactive inositol into chloroform extracts. The a p p a r e n t rma x computed from 5 different experiments with the H0 fraction was 10 ± 1.7 nmol min 1 per 10 l° infected cells, i.e., 0.023 nmol m i n - l (mg protein) 1. No significant incorporation of radioactive inositol into the homogenate H0 of uninfected erythrocytes could be detected (less than 0.16 nmol rain 1 per 10 l° cells) (n=2). The presence of detergent in the reaction mixture was a prerequisite for PI synthase activity. This activity from the solubilized fraction ($2) was gradually increased when the concentration of O G was increased from 4 to 30 m M where maximal P! synthase activity was obtained, which corresponds to a molar O G to C D P - D A G ratio of 150:1. Above this concentration, PI biosynthesis decreased sharply by 50% at 40 mM (data not shown). Assays of this activity were also carried out in the presence of Triton X-100 instead of OG. Maximal activity of the H0 fraction was observed in the presence of 20 mM Triton X-100 in the reaction mixture (17_+0.7 nmol min 1 per 10 l° infected cells) (n=3). Nevertheless, Triton X-100 was not used routinely since exposure of the enzyme to this detergent at 4°C resulted in gradual loss of the activity (80% after 1 h). On the other hand, OG-solubilized activity was much more stable, more than 80% of the enzyme activity being recovered after storage for 24 h at 0-4°C, and 100% after one month at - 8 0 ° C . Lastly, the addition of Triton X-100 in the incubation medium did not enhance OG-solubilized enzymatic activity.

3.2. Solubilization of PI synthase activity. PI synthase activity appeared to be membranebound since more than 90% of the activity observed in the crude hemolysate (H0) was recov-

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179-192

ered in the particulate fraction. PI synthase solubilization from the washed P. knowlesi-membrane fraction was carried out with O G according to previously described methods [17,20] which were adapted to the Plasmodium-enzyme. After 30 min solubilization with 40 mM OG, the H~ fraction was centrifuged at 100 000 × g for 30 min to obtain the solubilized fraction ($2) and pellet (PJ. The majority of the original PI synthase activity (88%) was recovered in the $2 fraction with a 23fold increase in specific activity (data not shown), indicating that O G treatment succeeded in solubilizing the membranous enzyme. The indespensability of the different factors present in the homogenization and solubilization steps is revealed in Table 1. In Plasmodium-infected erythrocytes, a great variety of proteinases has been described [21]. However, there was no clear effect of a proteinase inhibitor cocktail, at the usual concentrations (see Methods), on PI synthase activity in the homogenization and soluTable 1 Requirement for PI synthase activity in the enzyme preparation step from P. Knowlesi-infected erythrocytes PI synthase activity (% of control) Whole homogenate: medium A - antiproteinases 2-mercaptoethanol + 40 m M O G + 4 0 m M OG + 2 m M M n 2÷ + 3 0 m M Mg 2+

100 + 9 80_+ 10 70 + 5 22 + 2 67-t-4

Solubilization fraction:

100+2 103+3 95 ___5 0

medium B + antiproteinases -2-mercaptoethanol - M n 2+ and Mg 2+

The whole homogenate (H0) or the O G solubilized fraction ( S j were prepared from 5 x 109 infected erythrocytes as described in the Experimental Section with the specified modifications of medium A and B respectively. PI synthase activity was assayed in 20 #1 aliquots (corresponding to 3.3 x 107 and 108 infected cells for Ho and $2 respectively) under standard conditions. The results are means of 2 independent experiments carried out in triplicate and expressed as percentage activities recovered in Ho or $2 fractions when medium A and B were used (8.3 + 1.3 and 7.3 + 1 nmol min t per 10 I° infected cells respectively).

183

bilization steps. Nevertheless, proteinase inhibitors were routinely added during the homogenization step for caution. When the infected cells were lyzed in the absence of 2-mercaptoethanol, PI synthase activity decreased by 30% in the whole homogenate, but its omission at the solubilization step did not affect PI synthase activity. The direct addition of 40 mM O G at the first homogenization step led to a dramatic decrease (78%) in the activity that was largely antagonized by the simultaneous addition of bivalent ions to the medium A (Table 1). This was experimentally corroborated when the solubilization step was carried out without bivalent ions: the loss of activity was total and appeared irreversible since there was no recovery of enzyme activity when the OG-solubilized membranes were subsequently assayed in the presence of 100 mM Mg 2+ (standard assay conditions). This suggests the crucial involvement of bivalent ions to maintain the active form of PI synthase outside the membrane structure. Other detergents which have been used to solubilize PI synthase in yeast, i.e. Triton X-100 [17], or in rat liver, i.e. sodium cholate [22], were also examined for their solubilization effectiveness in P. knowlesi-infected erythrocytes. Sodium cholate added at 20 mM instead of OG in the reaction mixture medium led to moderate activity (3.7_+0.3 nmol rain 1 per 101° infected cells) of the enzyme in the homogenized H0 fraction. Furthermore, this detergent did not succeed in solubilizing, even at 47 raM, the activity and less than 5% was recovered as soluble activity ($2 fraction). By contrast, Triton X-100, although leading to a substantial activity at 20 mM in medium B, was no longer used since this detergent caused a drastic loss of PI synthase activity (see above). PI synthase, was routinely solubilized by 40 mM OG for 30 rain in the presence of Mn 2÷, Mg 2+ and glycerol to stabilize the enzyme. KC1 was also included in the solubilized buffer to increase the solubilizing effectiveness of OG. Indeed, the presence of salt is known to increase the solubilizing effectiveness of non-ionic detergents by decreasing their CMC and lowering the aggregation number of mixed micelles [23].

184

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179 192

3.3. Characteristics of solubilized Pl-synthase.

as phospholipase C. With this objective. P1 of"

Incorporation of [3H]inositol into PI was linear with time from 5 to at least 60 rain. PI synthase activity was also proportional to the amount of parasitized extract ($2), in the range of 0.5 2 x 108 infected cells (data not shown). PI-synthase activity was measured as a function of pH in the presence of 50 m M of either Tris-HC1 or Mops buffer. The activity was expressed between pH 6.5 and 9, with peak activity at p H 8 (data not shown). PI synthase activity absolutely required bivalent cations. The activity exhibited saturation kinetics when 3 m M inositol and 0.2 m M C D P D A G were held constant and the concentration of one of the two bivalent cations ( M r + or Mn 2+) was varied. The enzyme activities followed Michaelis-Menten kinetics with respect to Mn 2+ (Fig. I A). The Lineweaver-Burk representation shows an apparent Ks value of 0.1 mM. On the other hand, the saturation curve for Mg 2+ was not the Michaelis-Menten type. Activity increased with the Mg 2+ concentration without reaching a clear plateau up to 100 mM (Fig. I B). The differential effects of Mg 2~ and Mn 2 + used alone or in combination at saturating concentration are shown in Table 2. Mn 2 + was effective at much lower concentration than M f +, but at optimal concentrations the m a x i m u m activity obtained with 100 m M Mg 2+ was usually 2-fold greater than that obtained with 5 m M Mn 2+. The combination of both ions at their optimal concentrations, i.e. 5 mM Mn 2+ and 100 m M Mg 2 . , was not beneficial for PI-synthase activity, suggesting that the optimal concentration of 5 m M Mn 2+ prevented maximal activity sustained in the presence of 100 mM Mg 2+. However, further increase of the Mg 2~ concentration to 200 m M slightly increased the enzyme activity (Table 2). Ca 2 + (2 m M ) alone could not substitute Mg 2 ~ or Mn 2 ~. Furthermore, under optimal conditions (100 mM Mg 2 +), the addition of 2 mM Ca 2 + reduced PI synthase activity by 62% (Table 2). This inhibition could either have resulted from true inhibition or from stimulated breakdown of newlysynthesized PI. In this context, we looked for a potential Ca2+-sensitive degrading enzyme such

plasmodial membranes were prelabeled by incubating P. knowlesi-infected erythrocytes with 10 /~M [3H]inositol (0.8 Ci mmol 1) in medium I for 4 h at 37°C and the OG-solubilized membrane fraction ($2) was prepared as described in Materials and methods. Then $2 fractions were incubated under standard assay conditions for PI synthase, except for the absence or presence of 3 mM of non-radioactive inositol, and the additional presence of 2.5 mM Ca 2+ or its substitution by 5 mM EDTA. Regardless of the conditions,

R

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-4 [1/Mn

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'

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B

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40

60

80

100

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Fig. 1. Effect of Mn 2. and M f + on PI synthase activity. The solubilization fraction was prepared as described in Materials and methods except that the solubilization medium B was devoid of Mg 2÷ and contained 0.1 mM Mn2+; the assay medium contained 20 #1 extract in a final volume of 200 #1. Incorporation of 3 mM myo-[2-3H]inositol (4000 dpm nmol i) into PI was measured under standard conditions except for the indicated concentrations of Mn 2+ (A) or Mg 2+ (B). Insert corresponds to the Lineweaver-Burk representation of the saturation curve for Mn 2 ~.

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179-192 Table 2 Differential effect of bivalent ions on the solubilized PI synthase activity from P. knowlesi-infected erythrocytes Assays conditions

100 M m Mg z+ (standard) 5mM M n 2+ 1 0 0 m M Mg 2÷ + 5 m M M n 2+ 2 0 0 m M M g 2+ + 5 m M M n 2+ 2 m M Ca 2 + 100 m M Mg 2+ + 2 m M Ca 2+

PI synthase activity (% of control) 100_+6 56_+3

185

CMP. When aliquots of OG-solubilized fraction ($2) were incubated in the standard reaction mixture but with the additional presence of 3 mM CMP and 1 m M PI (sonicated preparations), the rate of incorporation of [3H]inositol into PI was reduced by about 50%. This reduction was not observed when only CMP was added (data not shown). These results could indicate that PI synthase obtained from P. knowlesi could also cat-

56+11 69.4+3 5 -+ 1 38___2

8 Assays of PI synthase activity were carried out with the solubilized fraction ($2) prepared as described in Materials and Methods except that M G 2 + was totally absent from medium B and the M n 2+ concentration was 1.0 m M (final M n 2+ concentration in the reaction mixture medium was 0.01 mM). Effects of Mg 2÷, M n 2+ and Ca 2+ at the indicated concentrations were measured in the presence o f 0.2 m M C D P - D A G . 100% of the activity corresponded to 9.3 nmol min -l per 10 l° infected cells.

changes in the level of prelabeled PI were never observed (result not shown). Simultaneously, PI synthesis was normally active as measured by the further addition of radioactive inositol to the prclabeled membrane fraction, but was reduced when 2.5 mM Ca 2+ was added to the reaction mixture, as already shown in Table 2. Experiments carried out under standard conditions using mixed micelle substrates of C D P - D A G and O G (which was held constant at 30 raM) showed that PI synthase activity was entirely dependent on the presence of C D P - D A G and inositol (Fig. 2). The saturation kinetics with myo-inositol or C D P - D A G showed Km values of 0.16_ 0.1 mM and 1+0.5 mM ( n = 3 ) respectively. Only C D P - D A G was inhibitory at high concentration (Fig. 2A). In this case, the apparent Vmax. obtained from the Lineweaver-Burk representation was undervalued due to the fact that C D P - D A G was an inhibitor at high concentration. Finally, we examined the effect of CMP on the incorporation of labeled inositol into PI. Indeed, PI synthase was also reported to be capable of catalyzing the reverse reaction [24], i.e. production of C D P - D A G and inositol from PI and

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[In0sit01] (mM) Fig. 2. Dependence of PI synthase on the concentration o f C D P - D A G and inositol. Incubations were carried out with the solubilized cell-free extract as described in Materials and methods except for the concentration of C D P - D A G (A) and inositol (B). Inserts show double reciprocal plots o f the initial velocity vs. substrate concentration. (A) C D P - D A G was varied while O G (30 raM) and [3H]inositol (3 raM, 4000 d p m n m o l - i ) were held constant. (B) Inositol was present at the indicated c o n c e n t r a t i o n s with specific activity r a n g i n g f r o m 900 to 48 000 d p m n m o l - 1 while C D P - D A G and O G were used at 0.2 m M and 30 m M respectively.

186

N. Elabbadi el al./Molecular and Biochemical Parasitolo~zy 63 ~1994) 179 192

alyze the reverse reaction, C M P + PI ~ C D P D A G + inositol, especially by using exogenous PI.

3.4. Purification of P I synthase.

Binding o f Pl synthase to the C D P - D A G - S e p h a r o s e resin was carried out with the $2 fraction [17] in m e d i u m B. W h e n the volume ratio o f OG-solubilized extract (at 5 x 109 cells per ml) to resin was maintained at 2:1, only 30% of the PI synthase activity remained in the supernatant fraction. 78% and 60% o f the activity were recovered in the supernatant when ratios of 30:1 and 8:1, respectively, were used. M o r e important, binding to the resin was completely prevented when 2 m M C D P - D A G were added to the $2 fraction prior its application to the resin, indicating a specific interaction o f PI synthase with the C D P - D A G moieties on the resin. Elution was first carried out with 1 4 m M C D P - D A G in medium B. Unfortunately, the enzyme activity recovered in the eluate was quite low (less than 5%). The recovery was not significantly improved under various conditions, such as 15 h elution, the presence o f 0.8 M hydroxylamine and 20 m M Triton X-100 [17] instead o f O G , omission of salts and elution at 30cC in the presence of 3 m M inositol. Effects o f E D T A , Ca 2~ or acid conditions were not examined since their use would cause drastic decreases o f P! synthase activity (see above). Potential stabilization o f the enzyme in the eluate was not obtained by the addition o f 5 mg m l - I BSA a n d / o r 5 mg ml r P L mixture (PC, PE, and PS at the ratio found in the P. knowlesi-infected erythrocytes, 4:3:l, respectively [3]) to medium B. The following experiments were aimed at determining whether PI synthase was retained on the resin or eventually eluted in an inactive (non-detectable) form by submitting the various samples to S D S - P A G E (Fig. 3). Eluates (lane 4) showed only one band which was clearly apparent with an apparent relative molecular mass (Mr) of 58 000. However, when C D P - D A G - S e p h a r o s e resin, which was used to interact with PI synthase, was extracted at 100°C for 3 min with 5% SDS, and the extract was analyzed by electrophoresis (lane 5), two bands were revealed, one intense b a n d with a Mr 58 000 and a second m u c h less

intense band migrating with an apparent Mr 50 000. Both o f these spots were also clearly present in the h o m o g e n a t e (H0) and the OG-solubilized extract ($2) before and after its contact with the affinity resin, However, their relative quantities were much lower in the H0 fraction (lane 1), $2 fraction (lane 2) and $2 fraction which was incubated with resin (lane 3). Since no enzymatic activity was recovered in the eluate, we could not clearly attribute either major or minor bands to the PI synthase enzyme.

1

ii!

2

;i!~

3

4

5

6

Mr

................

-116 --84 --58 --45 --36.5 m26.6

Fig. 3. SDS-polyacrylamide gel electrophoresis. Samples were prepared as described in Materials and methods. The various lanes corresponded to (1), H0 fraction (from 3.3 x 107 infected cells); (2) and (3), $2 fractions (10s infected cells) before and after incubation with the affinity resin, respectively; (4), resin eluate (2 x 108 infected cells); (5), heat-treated CDP-DAG Sepharose resin after its incubation with $2 fraction (2 x l0 s P. knowlesi-infected erythrocytes); (6), protein molecular weight standards are /:~-galactosidase (116 000), fructosc-6phosphate kinase (84 000), pyruvate kinase (58 000), ovalbumin (45 000), lactic dehydrogenase (36 500), and triose-phosphate isomerase (26 600).

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179-192

3.5. Labeling of PL and PPI of P. knowlesi-infected simian erythrocytes. Whole infected cells (ring and mature stages) were incubated for 1-4 h in a phosphate and inositol-free, saline glucose medium (medium II) in the presence of 32p or [3H]inositol (not shown). After 3 h, 32p was highly incorporated in PC and PE, followed by PIP2, PI and PIP, then by PA, PS and cardolipid, indicating the predominant labeling of PIP2 and PIP, although these PL are present in trace doses in living cells. However, infected erythrocytes stopped PL biosynthesis after 3 h and lost labeling of PIP2 ( - 3 6 % ) , PA and PS ( - 3 0 % ) and PIP ( - 1 3 % ) after 3-4 h. It should be noted that the reduction of 32p labeling occurred in PS and PA, which are metabolic intermediates [3], or PPI whose level is usually highly sensitive to the ATP level [5]. We also observed substantial incorporation of radioactive inositol into PI and its incorporation into PIP and PIP2 was weak and significant after only 3 h incubation. These incorporations can only reflect the parasite contribution since mature mammalian erythrocytes completely lack de novo synthesis of these PLs

187

cytes required correction for the activity of unparasitized cells present in each preparation (see Fig. 5). For example, PA labeling of mature infected cell preparations (at 41 h) and of control experiments were 203 520_+ 16 320 dpm and 20520__ 1080 dpm (2 h) -1 per 9 x 108 total cells, respectively. By subtracting the contribution of uninfected cells present in the infected suspension (18.5% parasitemia), incorporation into infected cells reached 106 dpm (2 h) -1 per 9 x 108 infected cells, i.e. 49-fold the incorporation observed in uninfected cells. The same calculations showed that the incorporation of 32p into PIP and PIP2 in infected erythrocytes (100%) would be 155- and 30-fold higher than in control erythrocytes. This indicates that 32p incorporation into PPI of infected cells was higher than into control erythrocytes, and that this can only be due to the parasite machinery.

A

~PE

~PIP

[1,2]. 3.6. Labeling of PL and PPI in P. falciparum-infected erythrocytes as a function of parasite maturation. The following experiments were conducted with P. falciparum-infected erythrocytes, twice synchronised by sorbitol treatment [10]. The infected suspensions were incubated at different stages of parasite maturation for 2 h in saline-glucose medium (medium II) with trace doses of 32p. Incorporation of 32p into PC, PE, PI and PS was null in control erythrocytes as expected (not shown), but quite substantial after Plasmodium infection (Fig. 4). The rates of incorporation remained relatively low during the first part of the cycle (ring form) but increased substantially after the twentieth hour of the cycle until the end of parasite maturation (schizont form). The rates of incorporation of 32p into PIP2, PIP and PA according to parasite maturation, were similar to those observed in other conventional PL, with a marked increase in the second half of the cycle except for PIP2, whose level plateaued after 33 h. However, labeling of infected erythro-

24

48 0 T I M E OF THE CYCLE (h)

24

48

Fig. 4. Biosynthesis of PL by P. falciparum-infected erythrocytes as a function of parasite maturation. A bulk of P. falciparum-infected erythrocytes was twice synchronized by sorbitol treatment and cultured in complete medium using the candlejar method. After the last schizogony, at the indicated time in the parasite cycle, 9 × 108 total erythrocyte aliquots were washed once with 0.9% NaCI and then incubated in 1 ml of medium II containing 250 #Ci 32p for 2 h at 37°C. Mean parasitemia was 18.5% and did not significantly change during the studied cycle. Incorporations of 32p into PIP, PIP2 and PA of control erythrocytes (treated and incubated as infected suspension, including sorbitol treatment) correspond to 0 h on the xaxis. Indicated dpm are per 9 x 108 cells and each value is the mean of 4 experiments (infected suspension) or 8 experiments (control).

188

N. Elabbadi et al./'Molecular and Biochemical Parasitology 63 (1994) 179 192

3.7. PL Labeling in P. falciparum-infected erythrocvtes during long incubations. Short incubations did not allow us to observe any appreciable entry of [3H]inositol or [3H]glycerol into PPI. In this context, clear evidence of de novo synthesis of PPI by Plasmodium was obtained from incubation of human erythrocytes, infected or not, in a medium providing quite good cellular viability (modified RMPI 1640 without inositol supplemented with 10% AB + serum) for durations of 2-20 h in the presence of radioactive phosphate and inositol. Under these conditions, control erythrocytes had only incorporated 32p into PIP2, PIP and PA (Fig. 5D). Isotopic equilibrium was reached after 15-20 h for PIP and PIP2, which corresponds to the ATP isotopic equilibrium time. PA equilibrated more slowly due to slower turnover and DAG-kinase [25]. On the other hand, in P. falciparum-infected erythrocyte suspensions, 32p incorporations into these 3 PLs were significantly higher, but above all accelerated after 15 h incubation, when the parasites matured (Fig. 5A). A similar incorporation pattern was obtained for PC, PE and PS (Fig. 5B). The most significant increase was observed for PIP2: in control cells, the 32p labeling of this PL did not significantly change between 12 h and 19.5 h (9400_+650 dpm) due to reaching isotopic equilibrium, whereas in infected preparations, labeling of PIP2 suddenly increased from 12400_+930 to 21200_+ 1700 dpm. When we restricted acceleration only to the infected erythrocytes, the increase in the PIP2, PIP and PA labeling was much more pronounced. In addition, infected P. falciparum suspensions showed quite measurable incorporation of [3H]inositol into PPI, but this appeared only after 12 h incubation when the parasite matured (Fig. 5C). This incorporation, which was null in control erythrocytes (results not shown), can only represent the parasite contribution. 3.8. Effect of Ca2+ on the accumulation of inositol phosphates into ionomycin treated infected cells. Ionomycin is a calcium ionophore which, by increasing intracellular Ca 2+, activates the PIP2 specific-phospholipase C [5]. To investigate the effect of Ca ~+ on the release of inositol phos-

phates from their parent precursors, we labeled infected erythrocytes with [3H]inositol and then washed them with medium III in the presence or absence of 10% serum for 2 h at room temperature to remove the residual precursor. Then, 20 mM LiCI were added to the incubation medium to inhibit the degradation of inositol phosphates, followed 20 min later by addition of the indicated concentration of ionomycin. LiC1 alone had no effect on any of the inositol phosphate basal levels (data not shown). Applied at the concentration range of 4-10 x 10 7 M for 15 min (Fig. 6B), ionomycin induced accumulation of IP3 (3fold), IP2 (2.5-fold) and IP (slightly increased). The elevated levels of IP3 and IP2 were already

A

to % infected / /

/

/

/

/

~ PI

C

10%

infected

PIP

2

4

PIP2/" / / / /

/.. //.,

/

2

/

/...,,: z/-j.i,

/

m

PIP

0

f

X

lO

E Q. "(3

B

10 °/° in fected

10

20 PC

D

100

2(

not infected

20-

PE

50

PlP~ PA

10 ~

IPS 10

20

PLP 0

10

20

INCUBATION TIME(h)

Fig. 5. PL labeling of control and P. falciparum-infected erythrocytes. 8 × 10s erythrocytes, 10% ring-infected (A,B,C) or non-infected (D), were incubated in 1.5 ml medium I supplemented with 10% AB + h u m a n serum, 20 #Ci 32p and 5 pCi [3H]inositol. Incubations were carried out using 35 m m petri dishes in a candle jar (see Materials and methods). For incubations longer than l0 h, incubation media were renewed with fresh m e d i u m at 9 h. Solid lines correspond to 32p labeling and dotted lines to [3H]inositol labeling. Indicated d p m are per 8 x l08 cells.

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 179-192

sensitive at the lowest ionomycin concentration examined (significant effect at 10 - 7 M). The resuits presented in Fig. 6A also show that no significant changes were detected in the level o f IP3, IP2 or IP when the serum was maintained t h r o u g h o u t the whole experiment, regardless o f the ionomycin concentration.

4. Discussion

There was a considerable increase in the total a m o u n t o f membranes and PLs a c c o m p a n y i n g

3

A

1

'

.serum

g

1

-'6

"1

-6

-5

2

0

-7

Log [ionomycin (M)]

Fig. 6. Accumulation of [3H]inositol phosphates following ionomycin treatment of P. falciparum erythrocytes. Infected erythrocytes (11.5% parasitemia, 66% trophozoite, 34% schizont) were incubated at 3% hematocrit in (inositol-free) medium III supplemented with 10% AB ÷ serum and 7.7 #Ci [3H]inositol ml -j using the candle-jar method. After 13 h at 37°C, cells were washed three times with medium 1II containing (+ serum (A)) or not (-serum (B)) 10% AB ÷ serum for 2 h at room temperature. Cells (2 x 108) were then bathed in 2 ml of fresh medium III in the presence or absence of serum and maintained at 37°C for the rest of the experiment. LiC1 (20 mM) was added 20 min before the indicated ionomycin concentrations and persued for 20 min. The amount of label present in IP (O), IP2 (A) and IP3 (&) is expressed as dpm (2 x 108 cells) -1. Indicated dpm are per 2 x 10s cells and each point represents the mean of 3 determinations of a representative experiment.

189

Plasmodium growth inside the host erythrocytes. PI, the precursor o f PPI, increase by a b o u t 15fold as c o m p a r e d to 4-6-fold for other PLs after infection [3]. O u r primary aim was to demonstrate de n o v o PI synthesis in parasitized erythrocytes by characterization o f PI synthase (EC 2.7.8.11), the enzyme controlling synthesis o f PI f r o m C D P D A G and inositol. The presence o f a detergent in the assay mixture was imperative for PI synthase activity. The maximal activity o f O G was observed at 30 m M , which is two-fold higher than its C M C [20]. The sharp decrease observed above this concentration could have been due to the substrate dilution in the O G - P L - m i x e d micelles. PI synthase activity was membrane-associated as in other animal cells, where it is generally associated with the endoplasmic reticulum [20,22,26], but also in some cases with the plasma m e m b r a n e [26]. In yeast [7], it has been located in mitochondrial, microsomal and plasma m e m b r a n e fractions. Subcellular localization o f PI synthase activity was not studied since there are presently no satisfactory fractionation procedures for Plasmodium-infected erythrocytes. We examined three detergents (cholate, Triton X-100 and O G ) which are frequently used for solubilization o f PI synthase activity. Cholate was unable to solubilize this activity whereas both Triton X-100 and O G were effective. Triton X-100 presented a m a j o r d r a w b a c k since the solubilized activity was dramatically unstable. Solubilization with O G for 30 min resulted in an almost complete recovery o f PI synthase activity. Interestingly, there was crucial stabilization o f the O G solubilized enzyme by the bivalent cations M g 2+ or M n 2÷ (Table 1), which are also required for the in vitro activity o f this enzyme. In m a n y respects, characteristics o f Plasmodium PI synthase resemble those o f other cell systems. The apparent Km for C D P - D A G and inositol were 0 . 1 6 + 0 . 1 m M and 1 ___0.5 m M respectively, within the range o f values reported for C D P - D A G (0.07-1.8 m M ) and inositol (0.1-4.6 m M ) in m a m malian sources [27,22,24] and in yeast [28]. The enzyme was highly sensitive to the M n 2÷ concentration, with interactions following Michaelis-Menten kinetics, with an apparent Ks o f

190

N. Elahbadi et al./Molecular and Biochemical ParasitoloKv 63 (1994j 179 192

0.1 mM (Fig. IA). In most animal tissues 1 5 mM Mn 2~ results in maximum activity [20,22,29]. Mg 2~ was 2-fold more effective than Mn 2+ to sustain PI synthase activity, but very high nonphysiological concentrations were required as already reported in some tissues [22,30]. Ca 2+ alone could not sustain PI synthase activity and reduced it when measured under optimal Mg 2+ concentrations (Table 2), This inhibition was also reported for some mammalian [29] and protozoan cells [30]. in our case, the inhibition could not have been due to Ca 2 ~-sensitive PI-degrading enzymes since this ion did not reduce the [3H]PI-prelabeled parasitic fraction incubated in the assay reactional mixture (see Results). Another possibility for PI synthase enzyme regulation is catalysis of the reverse reaction. Indeed, PI synthases from higher eukaryotic cells [20,24] are able to catalyse the reverse reaction forming inositol and C D P - D A G from CMP and PI. The decrease in the radiolabeled PI in the presence of both CMP and PI could indicate either inhibition of the enzyme or that PI synthase from P. knowlesi also catalyses the reverse reaction. No purification of protozoan PI synthase has yet been reported while microsome-associated PI synthase of yeast has been purified to near homogeneity using CDP-DAG-Sepharose affinity chromatography [17]. Obtention of substantial amounts of this enzyme from higher eukaryotic cells has been hampered by its instability in the presence of detergents [22]. Our results clearly indicate that CDP-DAG-dependent interactions of PI synthase with C D P - D A G sepharose lead to complete disappearance of the activity from the supernatant. However, elution in an active form was unsuccessful under various conditions. Based on the SDS-PAGE results, it remains to be determined whether the major protein band (Mr = 58000) found in the resin eluate represents PI synthase. The protein was highly concentrated on the affinity resin in comparison to samples H0 and $2 (Fig. 3) and its presence in the eluate was dependent on CDP-DAG. The reported Mr of PI synthase was 60000 in rat liver [22] and only 34000 in yeast [17]. The second minor protein band ( M r - 5 0 0 0 0 ) could also concern other CDPDAG-dependent enzymes which have already

been purified by CDP-DAG-Sepharose affinity chromatography such as PS synthase and phosphatidylglycerophosphate synthase [7]. The above results provide the first characterization of PI synthase, the enzyme involved in the intense de novo PI biosynthesis in Plasmodium-infected erythrocytes. We then searched for evidence that the PPI metabolism was also present in the parasite. However, investigations on the PP! cycle in Plasmodium were complicated by the fact that the mammalian host erythrocytes possess both kinases responsible for stepwise phosphorylation of PIP to PIP2 and their corresponding phosphomonoesterases, which convert PIP2 and PIP back into P1 [2]. Erythrocytes also synthesize PA from DAG by specific kinases [25]. Experiments with P. knowlesi-infected erythrocytes carried out in a medium without phosphate and inositol showed that labeling of PIP and PIP2 was intense with 32p and just detectable with [3H]inositol. These experiments were not completely conclusive due to the possible contribution of white cells and platelets if not completely removed during the cellulose treatment. This medium also did not permit satisfactory parasite viability as shown by the sharp decrease in labeling of intermediate metabolites (PS) and of the directly ATPrelated phosphorylated PL (PA and PPI) after 4 h incubation. The following experiments were thus carried out with P. falciparum cultured in vitro in human erythrocytes, eliminating contaminating cells, and allowing synchonization and long incubations in conditions that supported full parasite growth. Kinetics of 32p-incorporation into PLs as a function of parasite maturation showed that the incorporation of 32p into every PL was always dominant when the parasite matured (Fig. 4). The most interesting evidence of de novo biosynthesis of PIP and PIP2 from [3H]inositol was obtained after long incubations of P. Jalciparuminfected erythrocytes (Fig. 5). PPI are minor PLs of the plasma membrane which are broken down by a specific phospholipase C into two second messengers, IP3 which mobilizes Ca 2+ from intracellular stores and D A G which activates protein kinase C. Since the natural stimulus in Plasrnodium-infected erythrocytes is

N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994) 17~192

unknown, we artificially activated intracellular phosphoinositidase C by increasing the intracellular concentration of free Ca 2+ with the ionophore ionomycin [5]. Fig. 6 shows that a Ca2+-depen dent phosphoinositidase induced accumulation of labeled inositol phosphates in the presence of LiC1, which blocks the breakdown of inositol phosphates [5]. There was major accumulation of IP3, indicating that the primary action of calcium was to stimulate hydrolysis of PIP2, yielding DAG and IP3. By contrast, the continuous presence of serum cancelled the increase in inositol phosphate labeling. This was probably related to the fact that the PPI pool available to phospholipase C is very small in growing cells because of persistently stimulated PPI breakdown [31]. Growth factors in the serum are probably involved in regulation of intracellular inositol phosphate levels [32] but it is also possible that serum components can bind the ionophore. The present work clearly demonstrates that PI, a precursor of PPI, was biosynthesized from CDPDAG and myo-inositol catalyzed by PI synthase (EC 2.7.8.11). The de novo formation of inositol phosphates (i.e. IP3, IP2, and IP) and their parent lipids (i.e. PIP2, PIP, and PI) occurred in erythrocytes after plasmodial infection and was parasite specific since de novo synthesis of both PI and PPI does not occur in non-nucleated erythrocytes [1,2]. This PPI pathway activation was detected at the late stage of parasite development. Ionomycin, which increased the intracellular Ca 2+ concentration, induced release of neo-synthesized IP3 probably by activating PIP2-specific phospholipase C. These results demonstrate the presence and activation of a PPI cycle, which involved a functional inositol phosphate/DAG pathway in this parasitic protozoan. The presence and role of a PPI cycle and its associated secondary messengers in other protozoa is poorly documented. The high turnover of PI in protozoa (Trypanosoma cruzi) [33] suggested an important metabolic and/or regulation role for this PL and it was recently reported that T. cruzi contains an active inositol phosphate/DAG signalling pathway [34]. PPI hydrolysis during Plasmodium growth could have an extremely important physiological function. PPI hydrolysis at the ex-

191

flagellation step during zygote formation has been shown in P. falciparum malaria [35]. The fact that phorbol esters, potent activators of protein kinase C, stimulate gametocyte formation in culture of P. falciparum [36], indicates a potent role of the PPI cycle in the biology of Plasmodium parasites.

Acknowledgements This work was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (Grant 920556), the Institut National de la Sant~ et de la Recherche M6dicale (C.R.E. 910616), the Commission of the European Communities (No. TS3*-CT92.0084) and the 'Groupement de Recherche' CNRSDCSSA-DRET No. G 1077. We owe special thanks to B. Bayard for helpful discussion and skilled assistance.

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N. Elabbadi et al./Molecular and Biochemical Parasitology 63 (1994j 179 192

Parasitol. 65, 418-420. [11] Vial, J.H., Thuet, M.J. and Philippot, J.R. (1984) Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers. Biochim. Biophys. Acta. 795, 372 383. [12] Homewood, C.A. and Neame, K.D. (1976) A comparison of methods used for the removal of white cells from malaria-infected blood. Ann. Trop. Med. Parasitol. 70, 249 251. [13] Kutner. S., Breuer, W.V., Ginsburg, H., Aley, S.B. and Cabantchik, Z.I. (1985) Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium faleiparum. Association with parasite development. J. Cell. Physiol. 125, 521 527. [14] Rock, R.C., Standefer, J.C., Cook, R., T, Little, W. and Sprinz, M. (1971) Lipid composition of Plasmodium knowlesi membranes: comparison of parasites and microsomal subfractions with host rhesus erythrocytes membranes. Comp. Biochem. Phys. 38B, 425-437. [15] Schacht, J. (1981) Extraction and purification of polyphosphoinositides. Methods Enzymol. 72, 62f~631. [16] Berridge, M.J. (1983) Rapid accumulation of inositol triphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem. J. 212, 849-858. [17] Fischl, A.S. and Carman, G.M. (1983) Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase. J. Bacteriol. 154, 304-311. [18] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. [19] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248-254. [20] Parries, G.S. and H.-N., M. (1984) Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese. Biochemistry 23, 4785-4791. [21] Schr6vel, J., Deguercy, A., Mayer, R. and Monsigny, M. (1990) Proteases in malaria-infected red blood cells. Blood Cells 16, 563 584. [22] Takenawa, T. and Egawa, K. (1977) CDP-diglyceride: inositol transferase from rat liver. J. Biol. Chem. 252,

5419 -5423. [23] Kagawa, Y. (1972) Reconstitution of oxidative phosphorylation. Biochem. Biophys. Acta 265, 297 338. [24] Bleasdale, J.E., Wallis, P., MacDonald, P.C. and Johnston, J.M. (1979) Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue. Biochim. Biophys. Acta 575, 135 147. [25] Ferrel, J.E. and Huestis, W.H. (1984) Phosphoinositide metabolism and the morphology of human erythrocytes. J. Cell. Biol. 98, 1992 1998. [26] Imai, A. and Gershengorn, M.C. (1987) Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum. Nature 325, 726~728. [27] Ghalayini, A. and Eichberg, J. (1985) Purification of phosphatidylinositol synthase from rat brain by CDPdiacylglycerol affinity chromatography and properties of the purified enzyme. J. Neurochem. 44, 175 182. [28] Carman, G.M. and Fischl, A.S. (1992) Phosphatidylinositol synthase from yeast. Methods Enzymol. 209, 305 312. [29] Jungalwala, F.B., Freinkel, N. and Dawson, R.M.C. (1971) The metabolism of phosphatidylinositol in the thyroid gland of the pig. Biochem. J. 123, 19 33. [30] Daniels, C.F. and Palmer, F.B. St. C. (1980) Biosynthesis of phosphatidylinositol in Crithidia fasciculata. Biochim. Biophys. Acta 618, 263 272. [31] L'Allemain, G., Paris, S., Magnaldo, I. and Pouyss6gur, J. (1986) A-thrombin-induced inositol phosphate formation in GO-arrested and cycling hamster lung fibroblasts: Evidence for a protein kinase C-mediated desensitization response. J. Cell. Physiol. 129, 167 174. [32] Kevin, J.C.C., Hunydy, L. and Balla, T. (1991) Second messager derived from inositol lipids. J. Bioenerget. Biomembr. 23, 7 27. [33] Antunes, A. and Oliveira, M.M. (1981) Phospholipid metabolism in Trypanosoma cruzi-l. Phosphate moiety turnover. Comp. Biochem. Physiol. 70B, 327 330. [34] Docampo, R. and. P., O.P. 0991) The inositol phosphate/ diacylglycerol signalling pathway in Trypanosoma cruzi. Biochem. J. 275, 407 411. [35] Pett, M., Martin, S. and Schneider, 1. (1989) Phosphoinositide hydrolysis in P. falciparum malaria. FASEB J. 3, 17371. [36] Trager, W. and Gill, G.S. (1989) Plasmodium Jalciparum gametocyte formation in vitro: its stimulation by phorboldiesters and by 8-bromo cyclic adenosine monophosphate. J. Protozool. 36, 451-454.