Molecular and Biochemical Parasitology, 58 (1993) 123-134 123 ~ 1993 Elsevier Science Publishers B,V. All rights reserved. / 0166-6851/93/$06.00

MOLBIO 01922

Uptake and metabolism of S-adenosyl-L-methionine by Leishmania mexicana and Leishmania braziliensis promastigotes

Jos+ L. Avila and Mar ia A. Polegre lnstituto de Biomedicina, Caracas, Venezuela

Received 8 September 1992; accepted 16 November 1992)

Promastigotes of Leishmama mexicana and Leishrnania braziliensis incorporate S-adenosyl-L-[3H-methyl]methionine (AdoMet) against a concentration gradient through a saturable system. This concentrative uptake requires metabolic energy and is sensitive to temperature and sulfhydryl reagents such as N-ethyl maleimide. Intracellular AdoMet exchanges with external AdoMet. At steady state, unaltered ADoMet in the intracellular pool is at about a 1800-fold concentration in relation to that found in the external medium. Glucose, galactose and ribose did not stimulate uptake rates. Incorporated AdoMet goes into the soluble AdoMet pool, where a small fraction is metabolized, chiefly into methylthioadenosine, decarboxylated AdoMet and methanol. After a 60 min pulse the radioactivity associated with the [3H]AdoMet incorporated dissapears with a half-time of 2 h. Transmethylation reactions were analyzed following [3H]AdoMet incorporation. Fractionation experiments indicate that 45-62% and 30-42% of the radioactivity is incorporated into lipids and protein methyl esters respectively, with 5-14% present in the soluble pool of parasites. Sinefungin or its cyclic derivative (1 and 10 #g ml i) in the incubation medium produces 58% and 64% inhibition of AdoMet incorporation into Leishmania promastigotes. Most transmethylation reactions are inhibited, as there is a 50% decrease in the total radioactivity present in both the base-labile and lipidic fraction, with a parallel increase in the percentage of radioactivity in the soluble pool. Previous results give evidence of the importance of AdoMet in American LeLvhmania promastigote metabolism.

Key words: S-Adcnosyl-t.-methionine; Leishmania mexicana; Leishmania bra=iliensis; Uptake; Metabolism; Sinefungin

Introduction

S-adenosylmethionine (AdoMet) is a sub- strate or precursor in at least 4 different classes of enzymatic transfer reactions: its adenosyl group is transferred to tripolyphosphate in the reversal of AdoMet synthetase reaction, its 3-

Correspondence address: J.L. Avila, Instituto de Biomedicina, Apartado 4043, Caracas 1010A, Venezuela. Telephone: (582) 7823211 FAX: 582-8611258.

Abbreviations: AdoHcy, S-adenosylhomocysteine; AdoMet, S- adenosylmethionine; CCCP, carbonyl cyanide-m-chlorophenyl hydrazone; FBS, fetal bovine serum; HPLC, high pressure liquid chromatography; MEM, minimal essential medium; MET, methionine; MTA, 5'-methylthioadenosine; NEM, N- ethyl maleimide; PBSG, phosphate buffered saline-glucose; PCA, perchloric acid; PCMB, p.-chloromercuribenzoate; TCA, trichloroacetic acid.

amino-3-carboxyl group to special uridilate residues of certain bacterial tRNA, its amino- propyl group after decarboxylation in poly- amine biosynthesis and its methyl group to different classes of compounds including proteins, nucleic acids, lipids and small molecules such as biogenic amines [1,2].

It is generally accepted that eukaryotic cells have a rather limited permeability to AdoMet [3-6]. The role of AdoMet in the physiology of ieishmanial parasites pathogenic to man is not known, and although AdoMet is present in animal sera [7], we have found that it is not essential for continuous Leishmania growth (unpublished results). The processes involved in AdoMet uptake by kinetoplastid parasites have not yet been investigated. The present communication studies the AdoMet uptake and metabolism characteristics by Leishmania

124

mexicana and Leishmania hraziliensis promas- tigotes.

Materials and Methods

Materials. S-Adenosyl-L-[Methyl-3H]methion - ine (73-85 Ci mmol i) and L-[methyl-3H]meth - ionine (70 Ci mmol- i ) , L_[35S]methionine(1000 Ci mmol--I), [3H]proline (17 Ci mmol- I ) , [14C]inulin (5 mCi mmol - I ) and 3H20 (15 /~Ci ml-1) were purchased from Amersham (Ar- lington Heights, IL). Unlabeled AdoMet, S- adenosyl-L-homocysteine, methylthioadeno- sine, amino acids,nucleosides, nucleotides, cycloheximide, cycloleucine and sinefungin were purchased from Sigma (St. Louis, MO). Culture medium components including mini- mal essential medium (MEM), fetal bovine serum, vitamins were from Gibco.

Methods. Organisms and growth conditions. Leishmania spp. stock cultures were the same previously described [8]. Parasites were main- tained at 28°C on blood-agar medium overlaid with phosphate buffered saline supplemented with 1 mg ml ~ glucose (PBSG). They were subcultured every 7 days.

For routine experimental work, cells were cultivated in MEM-2.5% fetal bovine serum (FBS) as described previously [9]. Four days later cells (at the late-log phase of growth) were removed by centrifugation in an International PR-6 centrifuge at 1000 x g for 20 min at 4:'C and resuspended in PBSG.

Uptake experiments. AdoMet uptake was measured by the rapid filtration technique [10]. Routinely, freshly isolated promastigotes (99% motile) were resuspended in PBSG to a cell density of 15--25 x 10 ~'cell ml i. Aliquots of the cell suspension (0.2 ml in each well) were dispensed into tissue culture multi-well plate (Flow Laboratories, Inc, McLean, Va 22102). AdoMet was then added at a final concentra- tion of 3.75 /xCi well i and a specific radio- activity of 75 /xCi nmol -~. 4 h after the addition ol" radioisotope, wells were aspirated under vacuum and filtered through glass fiber

fihers (Enzo Diagnostics. Inc., New York. NY) and immediately washed with 2 ml of cold PBS. When the effect of certain substances on AdoMet uptake was studied, parasites were first preincubated with them for 2 h. AdoMet uptake was then initiated as previously described. Incubation time was 6 h at 28C. The filters with cells were transferred to minivials containing 2 ml of Bray's scintilla- tion fluid [11]. Subsequent radioactivity deter- mination was performed on a LKB-Wallac, model 1215 Rackbeta, liquid scintillation counter.

In some experiments, proline uptake mea- surements were carried out simultaneously as described by Zilberstein and Dwyer [12] in order to control Leishmania promastigote membrane function.

Protein determination. Protein was measured by the method of Lowry et al. [13] using bovine serum albumin as standard.

Measurement of cell volume. Cell volume of motile American Leishmania promastigotes resuspended in PBSG was measured at 28'C as described in Reference 14.

AnaO'sis o[" A~h~Met and AdoHcv hv IIPLC. Pellets of l,e&hmania promastigotes were extracted with 0.1 M perchloric acid (PCA) tbr 30 min at 4 C . These samples were centrifuged (10 000 x g for 2 min at 18'C) and the resultant supernatants were analyzed by HPLC according to Thong et al. [15]. The eluent was collected into fractions and aliquots were analyzed for radioactivity. The concen- tration of AdoMet and AdoHcy in lwishmania extract was determined from its absorbance relative to standards. Using AdoMet as an internal standard a 95% recovery of radio- activity was obtained.

Analysis of methyl-tran,~J'er reactions. At var- ioust imes after incubation with [3H]AdoMet, promastigotes (10 v cell m l - ~) were spun down from the medium and immediately homoge- nized in cold 10% trichloroacetic acid (TCA). Incubation medium was carefully evaporated

and residue resuspended in an aqueous liquid scintillation mixture (ACS II, Amersham Corp.) and counted for radioactivity.

3H-methylated macromolecules were preci- pitated from the TCA homogenate of promas- tigotes by centrifugation at 2500 x g for 10 min. The radioactive pellet, containing 3H- methylated proteins, lipids and nucleic acids, was washed with 1 ml of 10% TCA and repelleted by centrifugation. Except otherwise stated, the washed pellet was incubated for approximately 20 min at room temperature in 0.3 ml of 1.0 N NaOH until the pellet dissolved. Macromolecules were reprecipita- ted by the addition of 0.1 ml of cold 40% TCA followed by incubation for 30 min on ice and recentrifugation. The supernatant obtained after the centrifugation step was analyzed for [3H]methanoi derived from the hydrolysis of protein [3H]methyl esters using the dry filter paper assay [16]. The pellet was extracted with I ml chloroform-methanol 2:1 (v/v) for 15 min and centrifuged at 2500 x g for 10 min. Supernatants represented the lipid fraction, which was air-dried and solubilized in ACS II. The pellet was further extracted with 0.2 ml 5% TCA at 90°C for 30 min. The supernatant,

125

which give the nucleic acid fraction was air- dried and solubilized in ACS II.

All scintillation counting was done as described before for uptake experiments.

Measurement of lipids. Lipids were extracted with chloroform-methanol (2:1 v/v). Phospho- lipids were deacylated [17] and the water- soluble products separated by paper chroma- tography [18]. The three major components: glycerophosphorylcholine, glycerophosphory- lethanolamine and glycerophosphorylinositol were located by staining the standards channel with nynhydrin followed by acid-molybdate spray for phosphorus. Appropiate areas of an unstained channel were eluted with water for analysis. The lipid-soluble fraction remaining after the removal of water-soluble hydrolysis products was recovered and in some experi- ments analyzed on an acid-washed Florisil column [17]. Three fractions were eluted from the column: the fatty acid methyl ester fraction with chloroform-methanol-water (11 : 1:0.12 v/ v/v), the cyclic acetal and alkyl ether fraction with chloroform/methanol /water (6:1:0.07 v/ v/v) and the sphingomyelin fraction with chloroform/methanol (1:4 v/v).

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Fig. 1. (A) Time course of 3 S-adenosyl[ H-methyl]methionine incorporation in Leishmania mexicana promastigotes. (B) S- adenosylmethionine incorporation as function of the concentration of parasites in the incubation medium.

Results

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Fig. 2. The e x c h a n g e o f A m e r i c a n l.eishmania i n t r a c e l l u l a r [ 3 H ] A d o M e t w i t h e x t r a c e l l u l a r A d o M e t . U n l a b e l e d A d o -

M e t ( l m M ) was a d d e d a t 8.5 h as s h o w n by the a r r o w .

2 5 - -

Khzetics q[" AdoMet incorporation hTto Amer- ican l_x, ishmania promastigotes. As no differ- ence between L. mexicana and L. braziliensis promastigotes was detected in our experi- ments, we will use the term American

Leishmania to refer to both subspecies. A typical time course of AdoMet incorpora-

tion shows a linear rate up to 6 h (Figs. IA, and 2). Similar results were found when American Leishmania promastigote concentra- tion varied between 25 100 l~g protein m1-1 (Fig. I B). A plot of the rate of uptake as function of AdoMet concentration shows a typical saturation curve (Fig. 3A). This indicates the presence of a finite number of incorporation sites on the cell surface which are involved in uptake. Fig. 3B is a Line- weaver-Burk plot of the data shown in Fig. 3A. The incorporation system has a Km of 0.125 /tM and a Vm~,x of 24 pmol (mg protein)- h - I

We observed some variation in individual experiments in the rate of Adomet uptake, due perhaps to some substance from culture medium, as for example methionine, remain- ing in final parasite suspension.

As the intracellular concentration of Ado- Met in American Leishmania promastigote is 1.8 + 0.3 nmol mg-~ protein, it was estimated that the steady-state values represent an intra- cellular concentration about 1800 times higher

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Fig. 3. Leishmania braziliensis: the k ine t i c s o f S - a d e n o s y l - [ H - m e t h y l ] m e t h i o n i n e i n c o r p o r a t i o n . (A) R a t e o f u p t a k e as f u n c t i o n o f A d o M e t c o n c e n t r a t i o n . (B) A d o u b l e - r e c i p r o c a l p lo t o f the d a t a s h o w n in (A).

than that in the uptake reaction mixture (0.25 pmol p l - i ) . This is based on the fact that in PBSG American Leishmania promastigotes cell volume is 4 + 0.3 pl (mg protein)-1. A value of 1.8 nmol (mg cells)-1 represents a concen- tration of 0.45 nmol pl-1 (0.45 mM), while the external medium has a concentration of 0.25 pM.

On the other hand, proline uptake experi- ments revealed that uptake rate: 100-150 nmol (mg protein)-1 h - i was similar in American Leishmania promastigotes kept in PBSG at 28°C by 1,2,4 and 6 h, giving evidence for an adequate membrane function.

Effect o f sugars, amino acids and metabolic inhibitors on AdoMet incorporation by Amer- ican Leishmania promastigotes. In order to establish the specificity of the AdoMet carrier, experiments were carried out where the incorporation of labeled AdoMet by Amer- ican Leishmania promastigotes was followed in presence of other amino acids and AdoMet analogs as well in presence of adenosine, AMP and ATP. It was expected that affinity of other amino acids for the AdoMet carrier would be indicated by the inhibition of AdoMet incor-

TABLE I

Effect of several metabolic inhibitors on AdoMet uptake by American Leishrnania promastigotes

Substances added to Percent of control pre-incubation medium

None (control) 100 2 mM iodoacetate 5 I mM N-ethylmaleimide 28 2 mM cyanide 15 25 mM NaN3 8 2 mM 2,4-dinitrophenol 33 5 mM arsenite 9 2 mM fluoride 47 2 mM CCCP 26 4 mM amytal 63 1 mM PMSF 100 2 mM p-chloromercuribenzoate 40 1 mM cycloheximide 89 1 /~g ml ~ sinefungin 42 l0 ~g ml - t sinefungin 36 I ~g ml i cyclic sinefungin 60 10 ,ug ml - i cyclic sinefungin 52 1 mg ml ~ deoxyglucose 57

Except for 5 mM arsenite, promastigotes maintained > 9 0 % motile after 2 h preincubation at 28°C.

127

poration in presence of those amino acids. In general, among the several L-amino acids (assayed at 1 mM: glutamate, aspartate, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, proline, trypto- phan, glycine, serine, threonine, norleucine, lysine, cysteine, arginine, glutamine and aspar- agine) and nucleotides (AMP, ATP) only i mM methionine inhibited AdoMet uptake by more than 50% (results not shown) On the other hand, the AdoMet analogs: sinefungin and its cyclic derivative (10 pg ml - i) inhibited [3H-methyl]AdoMet incorporation into Amer- ican Leishmania promastigotes (Table I). However, addition of 7.5 ~M unlabeled AdoMet or 1 mM adenosine to parasites incorporating [3H-methyl]AdoMet resulted in a considerably reduced net influx of Adomet. The addition of unlabeled AdoMet to cells nearly saturated with [3H]AdoMet results in a slight exchange of intracellular radioactivity with the unlabeled amino acid outside (Fig. 2). Not exchangeable intracellular radioactivity may represent the fraction that is incorpora- ted into lipids, nucleic acids and proteins and the products of methionine metabolism which have no affinity for the AdoMet carrier(s) and therefore will not exchange with exogenous AdoMet.

Table I shows the effect of several metabolic poisons (respiratory inhibitors, uncouplers of oxidative phosphorylation, and sulfhydryl reagents). All compounds tested were inhibi- tory which suggest that metabolic energy is required for AdoMet uptake. Notably, I mM cycloheximide showed hardly any significant effect on AdoMet incorporation, suggesting that in American Leishmania promastigotes AdoMet is not involved in protein synthesis.

Interestingly 2 h absence of glucose in the pre-incubation medium did not modify Ado- Met incorporation, while other sugars (such as 1 mg m l - "ribose or galactose) did not increase AdoMet incorporation, although 1 mg ml - deoxyglucose inhibited in 43% AdoMet in- corporation.

AdoMet and AdoH~ T pools in American Leishmania promastigotes. There is only a

128

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Fig. 4. Incorporation of L-methionine into American Leishmania. Promastigotes were incubated with [me- thyl-3H]methionine (0.1 mM, 0.2 /~Ci /~mol - I ) and samples were withdrawn at intervals as indicated. C)---C),

total uptake; & - - A , cold-TCA-insoluble components.

short previous report of AdoMet in American Leishmania promastigotes [15], although these parasites contain methylated proteins such as calmodulin [19].

We have used cation exchange HPLC to

measure intracellular AdoMet and AdoHcy concentration in American Leishmania pro- mastigotes as described in Materials and Methods. We estimate that American Leishmania promastigotes contains 1.8 _+ 0.3 nmol AdoMet (mg protein)- i and 0.41 +_ 0.7 nmol AdoHcy (mg protein) '

Biosynthesis of AdoMet by American Leishma- nia promastigotes. In eukaryotic cells, Ado- Met is synthesized by a cytosolic synthetase which uses methionine and ATP as substrates [1,20]. When American Leishmania promasti- gores were incubated simultaneously with 35S- methionine and [methyl-3H]methionine, we found linear incorporation rates up to 20 min (Fig. 4). Both labels were incorporated into a radioactive peak which co-chromatographs with authentic AdoMet standards and con- tains both radioactive sulfur and tritium in the same ratio as the precursor methionine (Fig. 5). Since methionine could be directly incor- porated into proteins or enter the amino acid pool and be metabolized, the intracellular fate of the amino acid after entry was determined.

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Fig 5. Incorporation of radioactivity from isotopic methionine into acid-soluble molecules in Leishmania hraziliensis promastigotes. Parasites cultured for 4 days were incubated for 1 h in 0.5 ml of 2.5% FBS-methionine free-MEM containing

50 #Ci each [3~S]methionine (1000 Ci mmol - i ) and [methyl-3H]methionine (70 Ci m m o l i). 0 - - O , 35S; O---C). 3H.

As shown in Fig. 4, during the initial 20-min period almost all intracellular radioactivity is recovered by cold TCA extraction. A small amount of label appears in cold TCA-insoluble materials (macromolecular fraction).

The size of the AdoMet peak relative to that of methionine decreases when Leishmania promastigotes are labeled in presence of 25 mM cycloleucine, an inhibitor of AdoMet synthetase activity [20]. We have also ob- served a small peak of radioactivity which co- chromatographs with S-adenosylhomocysteine (AdoHcy), the expected end product of AdoMet-dependent transmethylation reac- tions [1,20]. Consistent with its structure, this peak contains the sulfur, but not tritium, radioactivity (Fig. 5). The size of this peak is about 20% the size of the AdoMet peak regardless of the incubation period, indicating that the AdoHcy produced during transmethy- lation reactions is rapidly hydrolyzed in Leishmania promastigotes. We have detected some radioactivity comigrating with a stan- dard of methylthioadenosine, a by product of polyamine synthesis.

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Fig. 6. Metabolism of S-adenosyl[3H-methyl]methionine in Leishmania mexicana promastigotes. Parasites were incu- bated with [3H]AdoMet for 30 min and at various times afterward, aliquots of cells were homogenized in 0.2 ml of cold 0.1 M PCA. The samples were processed and chromatographed as described in Materials and Methods. Once separated by HPLC, the fraction which comigrates with the AdoMet marker (fractions 70-80) was analyzed

for radioactivity.

129

Kinetics of AdoMet metabolism in American Leishmania promastigotes. We have used a similar HPLC ,procedure to monitor the metabolism of VH]AdoMet following a 60- min pulse incorporation into promastigotes. As shown in Fig. 5, AdoMet is converted into several acid-soluble species which elute from the cation exchange column before AdoMet, methylthioadenosine being a very important secondary metabolite. After a 60-min pulse the radioactivity associated with the incorporated [3H]AdoMet dissapears with a half-life of 2 h (Fig. 6). Using the kinetic constant derived from this data together with the pool data, we calculated the absolute rate of AdoMet utilization in American Leishmania promasti- gotes as 3.2 pmol h - ~. As we show below, our evidence indicates that the most of the AdoMet in Leishmania promastigotes is used for transmethylation reactions.

Transmethylation reactions in American Le&hmania promastigotes. To trace the fate of methyl groups incorporated as [3H]AdoMet in American Leishmania promastigotes, cells were fractionated as described in Materials and Methods. Our initial experiments indicated that during the 4-h incorporation period about 90% of the radioactivity was incorpo- rated into macromolecules The radioactivity recovered with TCA-precipitable material, i.e. macromolecules and lipids, was classified either as protein methyl esters or as other methylated macromolecules according to the base lability of the incorporated methyl group. The radioactivity recovered from the medium which was chromatographically distinct from AdoMet, contained volatile and non-volatile compounds. The volatile radioactivity was identified as [3H]methanol by its characteristic partitioning into organic solvents [16]. The non-volatile material consisted of methylthioa- denosine and decarboxylated AdoMet.

The kinetics of incorporation of radioactiv- ity from [3H]AdoMet into protein methyl esters (in base-labile linkages to macromole- cules), into base-stable linkages to macromo- lecules (in lipids and into nucleic acids) and into soluble pool is depicted graphically in Fig.

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Fig. 7. Kinetic analysis of transmethylation reactions in American Leishmania promastigotes. Parasites were incubated with [~H]AdoMet. At various times after incubation, 10 7 parasites were fractionated according to the procedure described under Materials and Methods. Radioactive products were classified as: (O), cold TCA-extractable material; (O) total protein methyl esters in control conditions and ( A ) in the presence of cycloheximide), (A) radioactivity in hot TCA-soluble fraction. (m) and in lipids. The results shown here represent the average of three experiments carried out on both L. mexicana and L.

hraziliensis promastigotes.

7. It is clear from the data that most radio- activity is incorporated into lipids (45-62%) and protein methyl esters (30-42%), with a lower percentage (5--14%) into the soluble pool of the parasites.

Notably, 30 min after the incubation period the radioactivity present in the cold TCA- soluble fraction represented only 5% of total parasitic radioactivity, this latter value increas- ing to reach a maximum (about 11-14%) at about 2 h incubation, and decreasing there- after. When using methionine as precursor, at 30 min most of the radioactivity is present in the cold TCA-soluble fraction, this indicates a slow conversion of methionine into AdoMet, radioactivity then being mainly incorporated into macromolecules and to a minor degree into other metabolites.

With regard to the radioactivity incorpo- rated into base-stable linkages with American Leishmania promastigote macromolecules, our results indicate that 45-62% of this radio- activity can be extracted with chloroform/ methanol 2:1, therefore they are considered

as lipids. A very much lower percentage, 1- 4%, was further extracted with 5°/,, TCA at 90<'C for 30 min, a procedure that afforded nucleic acid radioactivity.

Table II shows the distribution of radio- activity from the American Leishmania pro- mastigote lipid fractions. It can easily be seen that 81% of the radioactivity is associated with alkali-labile compounds (99% with glycero- phosphorylcholine and 1% with glyceropho- sphorylethanolamine) and 19% with alkali- stable compounds (95% with fatty acid methyl esters and 5% with cyclic acetals and alkyl ethers).

Inhibition by sinefungin and its cyclic derivative of trans-methylation reactions in American Leishmania promastigotes. As in a previous paper we described the strong inhibitory effect of sinefungin on American Leishmania protein carboxymethyl-transferase [8], we studied the effect of this drug and of its cyclic derivative on the uptake and kinetics of transmethylation reactions in these parasites. The results from

131

TABLE II

Distribution of radioactivity in fractions of the lipids directly obtained from the TCA-precipitable pellet from American Leishrnania promastigotes incubated for 4 h with [3H-methyl]AdoMet

Fractions Percent of lipid radioactivity

Total alkali labile 81 Fatty acid methyl esters 95 Cyclic acetals and alkyl ethers 4.9 Sphingomyelins < 0.1

Total alkali stable 19 Glycerophosphorylcholine 99 Glycerophosphorylethanolamine 0.9 Glycerophosphorylinositol < 0.1

these experiments indicate: (a) that 1 and 10 pg ml-1 sinefungin or its cyclic derivative in the incubation medium produce a 58% and 64% of inhibition of AdoMet incorporation into Leishmania promastigotes (Table I) the cyclic derivative being less effective than sinefungin, and (b) that most transmethylation reactions are inhibited, as there is a 50% decrease in the total radioactivity present in both the base- labile and lipidic fraction, with a parallel increase in the percentage of radioactivity in the soluble pool (Table III). These results can be easily explained: (a) by competition between sinefungin and AdoMet by same uptake site, (b) by sinefungin effectively inhibiting carbox- ylmethylation reaction, (measured by the decreased production of [3H]methanoi and protein [3H]methyl esters) as well as the transfer of methyl groups into base-stable linkages with macromolecules; (c) that sine-

fungin significantly increases the AdoMet soluble pool as consequence of inhibition of all transmethylation reactions (Table III). We believe that our present results give some evidence for all these possibilities.

Discussion

Results presented here show that American Leishmania promastigotes grown in culture are capable of accumulating AdoMet, against a 1800-fold concentration gradient across the cell membrane. The involvement of a carrier in AdoMet uptake is indicated by saturation kinetics, exchange of intracellular AdoMet with externally added AdoMet, and strong inhibition of uptake by the sulfhydryl reagents NEM, PCMB and iodoacetate. The concen- trative uptake of AdoMet is dependent on metabolic energy as indicated by its sensitivity to uncoupling agents (2,4-dinitrophenol, CCCP and arsenate) and to the respiratory inhibitors, KCN and azide. Surprisingly, addition or deletion of readily metabolizable energy sources such as glucose, ribose or galactose did not modify uptake rate, al- though a nonmetabolizable analog 2-deoxy- D-glucose and low temperatures (4°C) strongly inhibited AdoMet uptake rate (results not shown).

As AdoMet incorporation was strongly blocked by 1 mM methionine and adenosine, it could be hypothesized either that Adomet uptake occurs through methionine and adeno- sine carrier systems or that there is only one

TABLE Ill

Effect of sinefungin on uptake and incorporation of methyl groups from AdoMet in American Leishmania promastigotes

Control cells (pmol (mg prote in) - t ) Sinefungin-treated cells

(%) (%)

Soluble pool 17.02 + 5.27 13 + 2 55 + 17 Total incorporation into macromolecules 132.41 _+ 56.65 87 + 2 Nucleic acids i.64 + 0.22 2 + 0.7 4 + 2 Base-labile 51.92 + 18.31 40 + 3 19 + 4 Lipidic molecules 61.84 + 28.28 47 + 2 23 + 8

Exponentially growing promastigotes were preincubated for 6 h at 28°C in the presence of 10/~g m l - i sinefungin. Incubation was initiated adding to each well 3.75/~Ci of [3H-methyl]AdoMet and was for 4 h at 28°C.

132

carrier specific for AdoMet. Evidence for this latter hypothesis is the fact that while sinefungin and its cyclic analog strongly blocked AdoMet incorporation by Leishmania promastigotes, both drugs did not block [3H-methyl]-l~-methionine incorporation (results not shown).

The data presented here indicate that the uptake system for AdoMet in American Leishmania promastigotes largely resembles the amino acid uptake systems existing in prokaryotic and eukaryotic cells [10,21,22]. It is interesting however to note that in rat liver cell, AdoMet uptake rate was not significantly modified by addition of methionine, but it was by the presence of 1 mM 2,4-dinitrophenol [231.

Our results show that American Leishmania promastigotes contain a pool of intracellular AdoMet which is used for a variety of transmethylation reactions [24-26], this pool representing about 13 + 2% of total cellular AdoMet. In American Leishmania promasti- gotes, most methyl groups are transferred to lipids and then to proteins in that order. In lipids, 81% of the radioactivity is associated with alkali-labile compounds (99% of the methylation reaction is carried out on glycer- ophosphorylcholine and at much lower degree on glycerophosphorylethanolamine) and 19% with alkali-stable compounds (95% with fatty acid methyl esters). Gl~cerophosphorylinositol was not labeled when [ H-methyl]AdoMet was used as precursor, suggesting that no radio- activity appeared in the glycerol moieties of the phospholipids. The finding of radioactivity from [3H-methyl]AdoMet exclusively in glycer- ophosphorylcholine suggests that the typical methylation pathway of lecithin synthesis is active in American Leishmania promastigotes. When attempts were made to detect the presence of the mono- and dimethylated intermediates in the formation of phosphati- dylcholine from phosphatidylethanolamine, virtually all the radioactivity was recovered in the glycerophosphorylcholine peak. These data tend to indicate that the intermediates of the methylation pathway do not accumulate and are present in only trace quantities, if at all.

These latter results on American Leishmania promastigotes are similar to those reported in Crithidia .[asciculata [26] using [me- thyl-~4C]methionine as precursor and in strik- ing contrast with those found for Xenopus laevis oocytes [27].

Regarding proteins, our kinetic analysis indicates that protein methyl esters turn over rapidly in vivo. We have observed that the lbrmation of protein methyl esters in Leishmania promastigotes is unaffected by cycloheximide, making it unlikely that carbox- ylmethylation is occurring mainly on nascent peptidc chains or that methyl-accepting sites arise primarily from errors in amino acid incorporation during translation. Similar re- sults have been reported for X. laevis oocytes by O'Connor and Germain [27]. Although we have not identified the methylated residue(s) directly, our results suggest that the other major substrates for transmethylation in Leishmania promastigotes are basic amino acids in proteins. The function of protein carboxylmethylation reactions in eukaryotic cells has not been completely established, but it has been suggested that the enzyme initiates either the repair or metabolism of proteins which have been damaged by spontaneous racemization and/or deamidation processes [16,28]. Using sinefungin, a nonmetabolizable analog of AdoHcy [29], we have shown that about 40% of the AdoMet in American Leishmania promastigotes is utilized for trans- methylation reactions involving primarily macromolecular substrates as the percentage of radioactivity present in the soluble pool increases from 13 + 2 to 55 + 17%, with parallel inhibition of protein methyl ester and lipid methylations. Our results are thus some- what different from those of Phelouzat et al. [30]. These authors studying Leishmania donovani promastigotes found a total decrease in the percentage of 3H-methyl groups incor- porated into all cellular fractions, protein carboxylmethylations being the most inhibited transmethylation reactions (89%) tbllowed by protein-stable N-methylations (69%) and lipid methylations (45%), while our results show a similar percentage of inhibition of protein

carboxyl and lipid methylations. The differ- ences in sinefungin effects in Leishmania promastigotes could be due either to the different parasite spp. studied or to differences in the methodology used, as Phelouzat et al. [30] added sinefungin and [3H-methyl]AdoMet together to growing parasites and then incu- bated for 16 h at 28°C.

Acknowledgements

We thank Angela Avila for continuous maintenance of the several Leishmania iso- lates, Madeleine Robert-G6ro (Institut de Chimie des Substances Naturelles, C.N.R.S. 91190 Gif-sur-Yvette, France) for generous gift of sinefungin and its cyclic analog, and Francisco Rivero and Elba Avila for logistic help.

The investigation was supported by an EEC grant 'Science and Technology for Develop- ment', subprogramme 'Medicine, Health and Nutrition in the Tropics', Contract TS2-M- 2726-F and by CONICIT (Project SI-2113).

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