Intravacuolar proteolysis in Plasmodium falciparum digestive vacuoles is similar to intralysosomal proteolysis in mammalian cells

Biochimica etBiophysicaActa 926 (1987) 170-176

170

Elsevier BBA22833

I n t r a v a c u o l a r p r o t e o l y s i s in Plasmodium falciparum digestive v a c u o l e s is similar to i n t r a l y s o s o m a l p r o t e o l y s i s in m a m m a l i a n cells I n p y o C h o i a n d J o h n L. M e g o Department of Biology, University of Alabama, Tuscaloosa, AL 35487 (U.S.A.) (Received 13 May 1987)

Key words: Digestive vacuole; Proteolysis; Proton pump; (P. falciparum)

Hemoglobin of intact human erythrocytes was labelled with [t2sI]iodide and these cells were inoculated with late-stage cultures of Plasmodium falciparum. Subcellular parasite particulate material containing intact digestive vacuoles filled with 12SI-labelled hemoglobin was incubated in sucrose-buffer medium and degradation of labelled intravacuolar hemoglobin was measured by precipitation with trichloroacetic acid. Proteolysis was maximal at pH 5.0 or in the presence of MgATP at pH 8.0. The stimulatory effect of MgATP was probably due to energization of a proton pump activity as reported by others (Krogstad, D.J., Schlesinger, P.H. and Gluzman, I.Y. (1984) J. Cell Biol. 101, 2302-2309). Proteolysis was also inhibited by ionophores and antimalarials. These results suggest that P. falciparum digestive vacuoles have an ATP-dependent acidification mechanism similar to mammalian lysosomes but with some exceptions. The properties of this intravacuolar proteolysis were remarkably similar to intralysosomal proteolysis in mouse liver or kidney preparations.

Introduction

Intraerythrocytic malaria parasites take up host cell cytosol, which consists mostly of hemoglobin, and digest it in intracellular vacuoles [1-3]. Plasmodium falciparum digestive vacuoles have a low pH (4-6) and contain acid proteinases to degrade host cell hemoglobin [7,8]. The antimalarial drug chloroquine accumulates in digestive vacuoles of malaria parasites by virtue of its basic properties [9], and the extent of its accumulation is dependent on transvacuolar pH gradients [10]. Chloroquine also increases the pH in digestive vacuoles [6,9] and inhibits the degradation of hemoglobin within the vacuoles [11,12]. Thus both cytosol digestion and anti-

Correspondence: I. Choi, Department of Biology, University of Alabama, P.O. Box 1927, Tuscaloosa, AL 35487, U.S.A.

malarial accumulation within digestive vacuoles are dependent on intravacuolar pH. Therefore, an approach to the study of the mechanism of action of antimalarials may be to study their effects on digestive properties of malaria parasite digestive vacuoles. Present evidence suggests that parasite digestive vacuoles are analogous to mammalian lysosomes in terms of low pH and proteolytic activity [6,7,9,11,12]. Low pH in digestive vacuoles is probably maintained by a proton pumping system similar to that present in lysosomes [13-17]. Recently, Krogstad et al. [6] found that MgATP decreases the pH of parasite vesicles, but there is no direct evidence for the effects of pH and ATP on proteolytic activity in parasite digestive vacuoles. In this study, we have labelled the hemoglobin of intact erythrocytes with [125I]iodide. When these labelled cells were infected with P. falciparum, the

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

171 parasites ingested labelled hemoglobin. Labelled hemoglobin-filled digestive vacuoles obtained from these parasites by disruption in a French pressure cell and centrifugation degraded intravacuolar hemoglobin during incubation in appropriate media at 37 ° C. Properties of this intravacuolar digestion were similar to those noted in cell-free preparations of m a m m a l i a n lysosomes filled with t25I-labelled denatured serum albumin [13,14,18, 191. Materials and M e t h o d s

Carrier-free Na125I was purchased from I C N Radiochemicals, Irvine, CA. Iodo-beads (N-chlorobenzenesulfonamide-derivatized polystyrene beads) were purchased from Pierce Chemical Co., Rockford, IL. Rabbit anti-human erythrocyte antiserum was purchased from United States Biochemical Co., Cleveland, OH. A T P (disodium salt) and other nucleotides, ouabain, and oligomycin were purchased from Sigma Chemical Co., St. Louis, MO. Artemisinin and mefloquine were gifts from Dr. D. L. Klayman (Walter Reed A r m y Institute of Research, Washington). The G a m b i a n strain of P. falciparum was kindly provided by Dr. A.C. Vezza (University of Alabama, Birmingham). Parasites were maintained by serial passage in human erythrocytes. Infected erythrocytes containing 70-80% trophozoites and schizonts were collected by the gelatin flotation method [20]. Parasitemia was determined by microscopic inspection of Giemsa-stained thin blood smears. Carrier-free Na125I in 1 ml of Dulbecco's phosphate-buffered saline (buffer A, p H 7.4) was preincubated with 10 Iodo-beads at room temperature for 5 min as described by Markwell [21]. H u m a n erythrocytes (8.108 cells) were washed four times with ice-cold buffer A and then added to this mixture and incubated for 20 min. After incubation erythrocytes were decanted from Iodo-beads and washed four times with ice-cold buffer A. To measure the amount of radioactivity incorporated into the cytosol fraction, labelled intact erythrocytes were dialyzed against buffer A containing 5 m M glucose and 1 m M KI at 4 ° C overnight. Dialyzed erythrocytes were lyzed by suspension in 10 vol. of ice-cold 7.5 m M N a 2 H P O 4

( p H 7.4) for 1 h. Membranes and cytosol were separated by centrifugation for 2 h at 30 900 × g. Membranes were washed with 7.5 m M N a 2 H P O n until they became white (about five times). Cytosol was treated with trichloroacetic acid (5% ( w / v ) final concentration) and the precipitate was sedimented by centrifugation. Radioactivity in membranes and in precipitated cytosol fraction was counted in a Beckman G a m m a 4000 counter. The efficiency of this counter was 40% for ~25I. Although most of the radioactivity was incorporated into membranes, sufficient radioactivity was transferred into cytosol to label intracellular hemoglobin (Fig. 1). At about 20 min, the incorporation rate reached a m a x i m u m and no further increase was observed. Iodination reaction was dependent on the presence of Iodo-beads in the reaction tube (data not shown). In the absence of Iodo-beads, no detectable iodination was observed. The reaction mediated by Iodo-beads was also tempera-

(DPM × i0 -4)

(DPM x I0 -5)

1,2!

12 (6,0)

(6.0)

-~

1,0

>m-

10

(5,0

(5.0)

0,8 (4,0)

s (4,0)

O.6

6 (3,0)

(3.0)

4 (2,0)

0.4 (2.0)

[~

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2 (i,0)

(i,0 0

(0,0)

I

5

I

I

i0

i

20

r 30

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Fig. 1. Effect of incubation time on iodination of intact human erythrocyte. 0.5 ml of human erythrocytes (2.5-109), washed with ice-cold phosphate-buffered saline, were iodinated with 23 #Ci of 1251in the presence of 5 lodo-beads at room temperature for various times. 0.1 ml aliquots were taken and lyzed as described in Materials and Methods. Incorporation rates were calculated as the percentage of label incorporated of the total added radioactivity. Radioactivity (dpm) is indicated in parentheses. Radioactivity at zero time was subtracted from that obtained after 5, 10, 20 and 30 n'fin. Vertical bars denote S.E. of the mean of three separate determinations.

172 ture-dependent. Incorporated radioactivity at 4 ° C was about 30% of that at 25 ° C, but there was no difference between incorporated radioactivity at 2 5 ° C and that at 3 7 ° C (data not shown). To confirm the labelling of hemoglobin, cytosol was separated on a Sephadex G-100 column (1 × 57.5 cm) or by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate as described by Laemmli [22]. Gels were dried on a Pharmacia slab gel drier GSD-4 and exposed to K o d a k XAR5 X-ray film. Most of the cytosol radioactivity was found to be associated with hemoglobin, as expected (Fig. 2). 125I-labelled erythrocytes (3.9.109 cells) were inoculated with late-stage parasite-infected cells (1.8-109 cells, 70-80% parasitemia) enriched by the gelatin treatment. After 36 h of incubation, free parasites were obtained by immune lysis as described by Trager and Jensen [23] with some modifications. 10 ml of infected erythrocytes (25% hematocrit) were incubated at 37 ° C for 30 min in the presence of I ml sterile guinea pig serum and 1 ml rabbit anti-human erythrocyte antiserum (hemolytic titer of 1:3200). After removing the agglutinated plasma membranes by centrifugation at 270 × g for 1 min, the supernatant was centrifuged at 4300 × g for 10 min. Resulting precipitated free parasites were disintegrated by passage through a French pressure cell at 3000 psi (1 psi = 80.3 g / c m 2) in cold 0.25 M sucrose, 1 m M E D T A (pH 7.0). Disrupted parasites were centrifuged at 270 × g for 5 rain and the supernatant was centrifuged again at 30 900 × g for 10 min at 4° C. This fraction was used to assay the degradation of labelled hemoglobin. Labelled uninfected erythrocytes were treated in the same manner as controls. Radioactivity in uninfected controls was subtracted from the incorporated radioactivity in free parasites. Parasites (mostly trophozoites) incorporated about 1% of total erythrocyte radioactivity under these conditions. To assay proteolysis, sedimented parasite cell-free particulate fractions were suspended in media containing 0.25 M sucrose, 0.05 M 2-mercaptoethanol and 5 m M MgC12 unless otherwise indicated. All media were preincubated at 3 7 ° C and incubations were carried out at this temperature. Incubations were stopped by additions of 10% ( w / v ) trichloroacetic acid and the radioactivity was counted. Percentage

M.W.

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"~-- b a n d 3 - -

-~pas-1 ~ pas-4

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A

B

Fig. 2. Autoradiogram of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iodinated human erythrocytes. (A) Autoradiogram of detergent-solubifized, 125I-labelled erythrocytes. Labelled intact erythrocytes were solubilized in electrophoresis buffer containing 1% sodium dodecyl sulfate and electrophoresed on a 15% gel as described in Materials and Methods. The major membrane glycoproteins, band 3, PAS-1, and PAS-2 and the minor glycoproteins, PAS-3 and PAS-4, and hemoglobin are indicated as reported by Markwell and Fox [26]. (B) Autoradiogram of 125I-labellederythrocyte cytosol. Labelled intact erythrocytes were lyzed by suspension in 10 vol. of ice-cold 7.5 mM Na2HPO4 (pH 7.4). Cytosol was separated by centrifugation and electrophoresed on a 10% gel. (Molecular weight markers of (B): myosin, 205000; phosphorylase b, 97400; bovine serum albumin, 66000; ovalbumin, 45000; carbonic anhydrase, 29000; hemoglobin, 16000).

of acid-soluble radioactivity during incubation was calculated as described by Fouchier et al. [18]. All assays were carried out in duplicate or in triplicate. Results

Degradation of labelled hemoglobin in intact digestive vacuoles 270-30900 × g parasite particulate fractions obtained by passage through the French pressure

173

cell and centrifugation were incubated at various pH values with or without Triton X-100. If hemoglobin degradation occurred in intact digestive vacuoles in these preparations, the presence of detergent should disrupt membranes releasing vacuolar contents into the media, similarly to mouse liver lysosomes [24]. In the absence of 1% (v/v) Triton X-100, pH 5.0 buffer stimulated hemoglobin degradation approximately 40% (pH 8.0, 100; pH 5.0, 139.7 + 9.9). In the presence of detergent, Proteolysis was reduced 40% at both pH 8.0 and pH 5.0 (60.1 + 12.9 and 57.6 + 4.6 relative to controls of 100% without detergent at pH 8.0). Triton X-100 did not inhibit the proteolytic activity of crude cell-free parasite preparations (data not shown).

10

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4

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g A TP-stimulated intraoacuolar proteolysis At pH 8.0, intravacuolar proteolysis was inhibited approximately 30-40% and this inhibition was relieved by addition of ATP or pH 5.0 buffer (Fig. 3). Proteolysis was nearly linear up to 1.5 h and the stimulatory effect of ATP was relatively stable during this period. Linearity of hemoglobinase activity of P. falciparum extracts for approximately 90 min was also observed by

0 0

30

60

90

120

TIME(MIN) Fig. 3. Stimulation of proteolytic activity in crude digestive vacuole suspensions by A T P or p H 5.0 buffer. Proteolysis in crude digestive vacuoles was measured at 3 7 ° C after suspension in 0.25 M sucrose, 0.05 M 2-mercaptoethanol, 5 m M MgCI 2 and with additions as follows: O, 0.1 M Tris-acetate buffer (pH 5.0); O, 0.1 M Tris-acetate buffer (pH 8.0) and 2.5 m M ATP; zx, 0.1 M Tris-acetate buffer (pH 8.0); A, 0.1 M Tris-acetate buffer (pH 8.0) and 1 m M chloroquine. A T P was neutralized with K O H . Vertical bars denote S.E. of the mean of four separate determinations.

TABLE I E F F E C T S O F V A R I O U S N U C L E O T I D E S ON INTRAVAC U O L A R PROTEOLYSIS Crude digestive vacuole suspensions were incubated in 0.25 M sucrose, 0.05 M 2-mercaptoethanol, 5 m M MgC12 and 0.1 M Tris-acetate buffer (pH 8.0) at 3 7 ° C for 1.5 h with various nucleotides. Concentrations of nucleotides were 2.5 raM. Trichloroacetic acid-soluble radioactivities were determined as described in Materials and Methods and expressed as percentages of controls. Data represent m e a n s + S . E , of three separate experiments. Nucleotides

Acid soluble radioactivity (% of control)

N o addition (control) ATP UTP CTP GTP ADP AMP cAMP

100 183.6 ± 160.0 + 140.5+ 110.3+ 123.0+ 105.5 + 115.5+

17.0 15.0 8.8 9.7 1.0 0.5 0.5

Scheibel et al. [25]. Krogstad et al. [6] have also shown that the acidification of P. falciparum vesicles by ATP was stable for 2 h. Fig. 4 shows that the addition of ATP after 50 min incubation at pH 8.0 produced an increased rate of proteolysis. The effects of other nucleotides on proteolysis were tested (Table I). All nucleotide triphosphates tested except GTP showed stimulatory effects, with ATP being the most effective. ADP, AMP and cAMP were not effective. The stimulatory effects of ATP were maximal in the presence of MgC12 (Table II). Although some stimulation was obtained by ATP or MgC12 alone, these effects were less than ATP and Mg 2+ together. Krogstad et al. [6] also observed that the acidification of vesicles occurred in the presence of both ATP and MgC12.

174

Effects of some inhibitors and antimalarials on proteolysis

14

12

IO

8 NN

~ S =

4 2

20

40

60

80

i00

When the pH gradient across the digestive vacuole membrane was dissipated by protonophores, intravacuolar proteolysis was reduced (Table III). These protonophores abolished the stimulatory effect of ATP almost completely. Proteolysis in the vacuoles was insensitive to the (Na++ K+)-ATPase inhibitor, ouabain, and to the mitochondrial ATPase inhibitor, oligomycin. This suggests that the stimulatory effect of ATP at pH 8.0 was due to a restoration of the pH gradient across the membrane by activating an energy-dependent proton pump system. Proteolysis in vacuoles was also inhibited by various antimalarial drugs (Table IV). The most effective of these was mefloquine, and primaquine, an extraerythrocyte antimalarial, was least effective.

TIME(MIN) Fig. 4. Stimulation of the renewal of proteolysis by ATP after inhibition at pH 8.0. Crude digestive vacuole suspensions were incubated at 37 ° C in 0.25 M sucrose, 0.05 M 2mercaptoethanol, 5 mM MgC12 and 0.1 M Tris-acetate buffer (pH 8.0). After 50 rain, ATP was added (arrow). Vertical bars denote S.E. of the mean of three determinations.

Discussion

Markwell and Fox [26] have demonstrated that plasma membrane proteins of intact human

TABLE III EFFECTS OF INHIBITORS OF INTRAVACUOLAR PROTEOLYSIS

TABLE II EFFECTS OF D I V A L E N T CATIONS A N D THE STIMULATION OF I N T R A V A C U O L A R PROTEOLYSIS BY ATP Crude digestive vacuoles were preincubated in 0.25 M sucrose, 5 mM EDTA for 10 min on ice. Vacuoles were washed with ice-cold 0.25 M sucrose and resuspended in 0.25 M sucrose, 0.05 M 2-mercaptoethanol, 0.1 M Tris-acetate buffer (pH 8.0) with or without 10 mM cation. Incubations were carried out at 37 ° C for 2 h. The concentration of ATP was 2.5 mM. Trichloroacetic acid-soluble radioactivities were expressed as percentages of control. Data represents means_+S.E, of three separate experiments. Cations and ATP

Acid-soluble radioactivity (% of control)

No addition (control) ATP Mg 2+ MgATP Ca 2+ CaATP MnATP

100 111.5 + 12.8 140.9_+ 14.6 183.6 _+17.0 133.5_+ 5.6 134.1 + 14.6 100.0_+ 1.5

All samples contained 0.25 M sucrose, 0.05 M 2mercaptoethanol, 5 mM MgC12 and 0.1 M Tris-acetate buffer (pH 8.0). 2.5 mM ATP was added to all samples except pH 8.0 buffer only. A 2,4-dinitrophenol stock solution was prepared by dissolving in 95% ethanol and equivalent concentrations of ethanol were added to all samples. The nigericin experiment was carried out as described by Mego [29]. Incubations were carried out at 37 ° C for 2 h and trichloroacetic acid-soluble radioactivities were expressed as percentage inhibitions of control. Data represent means _+S.E. of three separate experiments, except for the nigericin experiment. Inhibitors

Percent inhibition

pH 8.0 + ATP pH 8.0 buffer only 2 / t M A23187 5 mM 2,4-dinitrophenol 50 # g / m l oligomycin 100/~M ouabain 1 ~tM nigericin 100 ~M carbonylcyanide po trifluoromethoxyphenylhydrazone

(control) 31.5 + 5.0 29.7_+5.2 20.5 _+7.5 3.0_+ 6.1 10.8 ___1.7 28.6 34.2 _+4.3

175 T A B L E IV EFFECTS O F A N T I M A L A R I A L S A N D NH,tC1 O N INTRAVACUOLAR PROTEOLYSIS All samples c o n t a i n e d 0.25 M sucrose, 0.05 M 2mercaptoethanol, 5 m M MgC12 and 0.1 M Tris-acetate buffer (pH 8.0). 2.5 m M A T P was added to all samples except p H 8.0 buffer only. The concentrations of antimalarials and NHaC1 were 1.0 mM. Incubations were carried out at 37 ° C for 2 h and trichloroacetic acid-soluble radioactivities were expressed as percentage inhibitions of control. Data represent m e a n s + S.E. of four separate experiments. Antimalarials and NH4C1

Percent inhibition

p H 8.0 + A T P p H 8.0 buffer only Quinine Chloroquine Mefloquine Artemisinin Primaquine Quinacrine NHaC1

(control) 31.5 + 5.0 41.2 + 2.7 78.7 + 0.3 87.8 + 9.9 39.0 + 1.3 26.2 + 1.8 70.3 + 3.1 48.8 ___2.4

erythrocytes may be labelled with 1251 using Iodogen (1,3,4,6-tetrachloro-3 a,6 a-diphenylglycoluril). These workers showed that some iodination of soluble cytosolic protein occurred under the conditions used. In the present studies, we have been able to label the hemoglobin of intact human erythrocytes using a similar technique. We have also labelled the cytosolic proteins of rat erythrocyte, Escherichia coli and Euglena gracilis (data not shown). An interesting observation during these studies was that a significantly higher level of cytosolic labelling occurred, relative to isotopic incorporation into membrane proteins, with rat erythrocytes than with human cells. Others have also observed some differences in incorporation rates into cytosolic proteins of human erythrocytes [26] and sheep erythrocytes [27]. In the studies reported in this paper, parasites apparently free of erythrocyte membranes were obtained by immune lysis [28]. If saponin or hypotonicity was used to rupture parasite-containing erythrocytes, the degradation of 125I-labelled hemoglobin was not observed in cell-free preparations obtained by pressure-cell disintegration. The reason for this is not clear, but most likely these treatments somehow damaged the digestive vacuoles or caused an inhibition of intravacuolar

proteinase. Since radioactivity was sedimentable by centrifugation when the saponin or osmotic methods were used to disrupt erythrocytes, digestive vacuoles presumably remained intact after French pressure cell disintegration. When cell-free preparations were treated with the detergent Triton X-100, radioactivity was not sedimented by centrifugation and rates of hemoglobin degradation were greatly reduced, suggesting that vacuolar contents were diluted in the media. Rates of hemoglobin degradation in crude digestive vacuole preparations were affected by pH (Fig. 3). The inhibitory effect of pH 8.0 buffer was relieved by pH 5.0 buffer or by MgATP. The stimulatory effect of ATP was most likely due to energization of a proton pump similar to that observed in crude mammalian lysosome preparations degrading 125I-labelled albumin [13,14,19]. Protonophores abolish the stimulatory effect of ATP on intralysosomal proteolysis in mouse kidney or liver lysosomes [19,29]. The inhibitory effects of protonophores on the stimulation of intravacuolar proteolysis by ATP were also observed in Plasmodium digestive vacuoles (Table III). Krogstad et al. [6] have shown that ATP decreases the pH in Plasmodium digestive vacuoles of infected cells. This has been observed in lysosomes [15,16]. Thus the properties of intravacuolar degradation of 125I-labelled hemoglobin observed in the studies reported in this communication are remarkably similar to those in crude suspensions of lysosomes from liver and kidney [13,14] or rat thyroid glands [18]. However, some significant differences were noted. For example, Plasmodium digestive vacuoles appear to be considerably more stable than mammalian lysosomes. In rat liver lysosomes, purine nucleotides (ITP, GTP) stimulated proteolysis and pyrimidine nucleotides were without effect [13]. GTP was entirely without effect as a stimulant of hemoglobin degradation in Plasmodium digestive vacuoles, but UTP was as effective as ATP. Thus the proton pump system of Plasmodium digestive vacuoles must be somewhat different from that in the lysosome membrane. It appears unlikely that the presence of some intact parasites in cell-free suspensions of the digestive vacuoles used in these experiments may account for the observed differences in nucleotide triphosphate stimulation, since we have been unable to

176 d e t e c t s i g n i f i c a n t p r o t e o l y t i c rates in these o r g a n i s m s b y t r i c h l o r o a c e t i c acid p r e c i p i t a t i o n . F u r t h e r m o r e , no d e g r a d a t i o n of p a r t i c u l a t e - a s s o c i a t e d labelled h e m o g l o b i n o c c u r r e d in pellets resulting f r o m p r e l i m i n a r y 270 × g (5 min) centrifug a t i o n of pressure-cell h o m o g e n a t e s . H o m e w o o d et al. [9] originally m a d e the suggestion that a n t i m a l a r i a l drugs a c c u m u l a t e w i t h i n digestive vacuoles where they raise int r a v a c u o l a r p H a n d t h e r e b y inhibit p r o t e i n a s e activity. This h y p o t h e s i s has b e e n p r o v e n b y the w o r k of K r o g s t a d et al. [6] in which direct p H m e a s u r e m e n t was m a d e b y fluorescence c h a n g e s using the t e c h n i q u e of O h k u m a a n d Poole [30]. In these studies, a n t i m a l a r i a l s a n d NH4C1 i n c r e a s e d i n t r a v a c u o l a r p H a n d i n h i b i t e d p a r a s i t e growth. Z a r c h i n et al. [12] also d e m o n s t r a t e d that c h l o r o quine a n d NH4C1 i n h i b i t e d a m i n o acid p r o d u c tion from host c y t o s o l p r o t e i n d e g r a d a t i o n . Antimalarials inhibited intravacuolar degradation of h e m o g l o b i n in the p r e s e n t studies. C h l o r o quine inhibited intravacuolar proteolysis nearly 80% at 1 m M c o n c e n t r a t i o n ( T a b l e IV) a n d m e f l o q u i n e was even m o r e effective. T h e degree of i n h i b i t i o n b y the a n t i m a l a r i a l s tested in these e x p e r i m e n t s c o r r e s p o n d e d closely to the conc e n t r a t i o n s r e q u i r e d to raise i n t r a v a c u o l a r p H b y 0.3-0.5 p H units o b s e r v e d b y K r o g s t a d et al. [6]. F o r example, the o r d e r of increasing effectiveness in raising p H was m e f l o q u i n e > c h l o r o q u i n e > quinine, which c o r r e s p o n d s e x a c t l y with degrees of inhibition of intravacuolar hemoglobin d e g r a d a t i o n ( T a b l e IV). This suggests that i n h i b i tory effects of these drugs reflect i n t r a v a c u o l a r c o n c e n t r a t i o n s a n d / o r relative abilities to raise i n t r a v a c u o l a r p H . G y a n g et al. [7] were u n a b l e to d e m o n s t r a t e an i n h i b i t i o n of P. falciparum p e p t i dase activity at 10 m M c h i o r o q u i n e c o n c e n t r a t i o n , a l t h o u g h these w o r k e r s n o t e d a c o m p l e t e inhibitory effect at 33.4 m M c h l o r o q u i n e at p H 6.0. T h e i n h i b i t o r y effects of 1 m M c o n c e n t r a t i o n s of antim a l a r i a l s tested in suspensions of i n t a c t digestive vacuoles ( T a b l e IV) m u s t t h e r e f o r e have b e e n d u e to extensive a c c u m u l a t i o n of the d r u g within the vacuoles, as p r o p o s e d b y H o m e w o o d et al. [9]. T h e a c c u m u l a t i o n of c h l o r o q u i n e within l y s o s o m e s [31] a n d p a r a s i t e digestive vacuoles [10] has also b e e n demonstrated.

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