EXPERIMENTAL
PARASITOLOGY
Plasmodium
43,
(1977)
knowlesi: In Vitro Biosynthesis and Thymidylic Acid
CRAIG C. SMITH,~ Dicision
248-254
of Medicinal
GEXALD Chemistry, Washington,
(Accepted
for
J, MCCORMICK, Walter D.C. publication
Reed 20012
of Methionine
AND CRAIG J. CANFIELD Army U.S.A.
13 May
Institute
of Research,
1977)
C. C., MCCORMICK, G. J., AND CANFIELD, C. J. 1977. Plas-modium ,&wl.&: biosynthesis of methionine and thymidylic acid. Experimental Parasitology 43, Mcthionine and thymidylic acid biosyntheses were studied to investigate methyl group sources in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with L-[3-‘Clserine, L-[ring-2-l”C]histidine, [“Clformaldehyde, [m&hyGYJcholine, [methyl-‘Clbetaine, [methyL%]sarosine, and 5-[formyE “C] formyltetrahydrofolic acid. Thin layer chromatography of acid hydrolysates of washed erythrocytcs showed that only L-[3-14C]serine was utilized as a methyl donor. In cultures utilizing L-[3-%]serine and L-13”S]homocysteine, the results of studies with pyrimethamine and 5fluoroorotic acid were consistent with their modes of action. An investigation of related folate metabolism revealed that large increases in thymidylic acid synthesis occurred with the addition of p-aminobenzoic, dihydrofolic, and tetrahydrofolic acids, and smaller increases with the addition of folic and diglutamylfolic acids to cultures containing L-[3-“Cl serine. With the exception of tetrahydrofolic acid, which had no apparent effect, all of the previously mentioned compounds induced a decrease in methionine synthesis, attributed to repression by methionine and its metabolic end products. Experiments utilizing L-["S] homocysteine produced small to moderate increases in methionine levels upon addition of folic, tetrahydrofolic, and methyltetrahydrofolic acids, presumably due to increased homocysteine levels, compensating for repressed cystathionase (EC 4.4.1.8) activity. INDEX DESCRIPTORS: Plasmodium knowlesi; Parasitic protozoa; Malaria; Methionine r.-[ring-2-14C]Histidine; [14C]Formaldebiosynthesis; L-[3-%]Serine; L-[YSlHomocysteine; hyde; [methyl-‘“C]Sarcosine; [methyZ-14C]Betaine; [methyZ-14C]Choline; 5-[formyl-‘“C]Formyltetrahydrofolic acid; Tetrahydrofolic acid; Dihydrofolic acid; Folic acid; p-Aminobenzoic acid; Diglutamyl folic acid; S-Methyltetrahydrofolic acid; 5-Fluoroorotic acid; Pyrimethamine; Rhesus monkey; Macacca mdutta; in citro culture; S-Adenosyl methionine; Methionyl tRNA. SMITH,
In vitro 248-254.
The association of folate metabolism and the biosynthesis of methionine and thymidylic acid is well established in mammals and microorganisms (Stokstad and Koch 1967; Blakely 1969). Thymidylic 1 Present address: Section on Adult Psychiatry Branch, National Mental Health, Bethesda, Maryland
Biochemistry, Institute of 20014, U.S.A.
acid is formed by transfer of the methylene group of 5,10-methylenetetrahydrofolic acid to deoxyuridylic acid by the action of thymidylate synthetase (this enzyme has no EC number). After the conversion of 5,10-methylenetetrahydrofolic acid to 5methyltetrahydrofolic acid by 5,10-methylenetetrahydrofolic acid reductase (EC 1.1.1.68), methionine is produced by donation of the methyl group to homo-
218 Copyright All rights
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
ISSN
0014
4894
Phmodium
knowlesi:
METHIONINE
cysteine by action of tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13) or tetrahydropteroyltriglutamate methyltransferase (EC 2.1.1.4). The initial 5,10methylenetetrahydrofolic acid may be the product of several mechanisms: reduction of 5,10-methenyltetrahydrofolic acid by 5,10-methylenetetrahydrofolic acid dehydrogenase (EC 1.5.1.5), reaction of serine and tetrahydrofolic acid by serine hydroxymethyltransferase (EC 2.1.2.1), and nonenzymatic reaction of formaldehyde and tetrahydrofolic acid (Blakely, Ramasastri and McDougall 1963). There is limited information concerning the presence of these reactions in malaria parasites. Thymidylate synthetase was demonstrated in Plasmodium lophurae by Walsh and Sherman ( RX%), in P. berghei by Reid and Friedkin ( 1973), and in P. chabaudi by Walter and Konigk ( 1971). Platzer (1972) demonstrated the presence of serine hydroxymethyl transferase in P. Zophurae and reported that lo-formyltetrahydrofolate synthetase (EC 6.3.4.3) and 5,10-methylenetetrahydrofolic acid dehydrogenase apparently were absent in this malaria parasite. Evidence of biosynthesis of methionine by P. berghei was obtained by Langer, Phisphumvidi, Jiamperpoon, and Weidhorn ( 1969), and Smith, McCormick, and Canfield (1976) observed biosynthesis of methionine and thymidylic acid by P. knowlesi; the methyl groups were derived from serine. This report describes a study of the association of folates and biosynthesis of methionine and thymidylic acid in P. knowlesi-infected blood in vitro. A survey was made of compounds which are sources of methyl groups in other organisms for possible utilization by this parasite. Evidence of relationship between the biosynthesis of methionine and thymidylic acid and the metabolism of folates was sought by observation of effects of folate and 5intermediates, pyrimethamine, fluoroorotic acid on the appearance in
AND THYMIDYLIC
ACXD
249
methionine and thymidylic acid of radioactivity from L-[3-lkC]serine and L-[““S] homocysteine. MATERIALS
AND METHODS
labeled compounds Radioisotopically used were L-[3-14C]serine (specific activity 56.5 mCi/mmole, L- [YS] homocysteine thiolactone (specific activity 6.1 or 8.2 mCi/ mmole ), and L- [ring-2-14C] histidine ( specific activity 55 mCi/mmole), obtained from AmershamJSearle, [‘Cl formaldehyde ( specific activity 59.2 mCi/mmole), [methyl-W] sarcosine (specific activity 3.86 mCi/mmole, [methy F’C] betaine ( specific activity 2.60 mCi/mmole), and [m&Z‘“Cl choline (specific activity 8.68 mCi/ mmole), obtained from New England Nuclear, and 5- [formyZ-14C]formyltetrahydrofolic acid (folinic acid) (specific activity 2.73 mCi/mmole) obtained from Monsanto Research Corporation. The specific activity of L- [ 3-“C] serine was adjusted to 28.8 mCi/mmole by addition of nonradioactive L-serine. L- [ 731 HOmocysteine thiolactone was supplemented with nonradioactive L-homocysteine, hydrolyzed in 1 N HCI for 24 hr, and neutralized with 1 N NaOH; the final specific activity was 0.37 mCi/mmole. The following quantities of radioactive compounds were added to cultivation tubes: L- [3-lC]serine, 0.7 pmole (20 &i); L-[35S]homocysteine, 54 pmoles (20 &i); L- [ring-2-l%] histidine, 0.18 pmole (10 &i); [14C]formaldehyde, 0.17 mole (10 &i); [methyZ-14C]sarcosine, 2.6 qoles (10 &i); [methyZ-14C] -betaine, 3.8 pmoles ( 10 &i); [ m.ethyZ-14C]choline, 1.15 wales ( 10 &i) ; and 5-[formyZ-14CJformyltetrahydrofolic acid, 1.5 eoles (4.1 &i). When used, tetrahydrofolic acid, dihydrofolic acid, folic acid, diglutamylfolic acid, p-aminobenzoic acid, and 5-fluoroerotic acid were present at a 0.01 mM con5-methyltetrahydrofolic acid centration, was 2.5 x 10m3mM, and pyrimethamine -.
250
SMITH,
MCCORMICK,
was 2 x 1O-5 m&f. All solutions were adjusted to pH 7.6 with 0.1 N NaOH. Procedures employed for cultivation in vitro and analysis were as reported in detail previously (Smith, McCormick, and Canfield 1976). Cultivation was done in Eagle’s minimum essential medium without folic acid, methionine, serine, or homocysteine, but supplemented with fetal bovine serum. The fetal bovine serum contributed 0.145 pmole of serine and 0.038 pmole of methionine per culture tube; homocysteine was not detected. Blood was taken from Plasmodium knowlesi-infected rhesus monkeys (Macacca mulatta) when 20-2570 of the erythrocytes contained a synchronous population of young trophozoites. The blood cells were washed, suspended (15% v/v) in medium, and 0.2ml aliquots were added to cultivation tubes. After incubation for 17 hr, blood cells were packed by centrifugation at 7006 for 15 min at 3-5°C washed three times with cold medium, and hydrolyzed in 6 N WC1 for 24 hr at 90°C. The hydrolysates were evaporated to dryness and soluble products were extracted into final volumes of 0.5 ml of 20% isopropyl alcohol in water. Aliquots (12 PI) were applied to silica gel thin layer chromatography plates and chromatotographic development was accomplished with a 4: 1: 1 mixture of 1-butanol, glacial acetic acid, and distilled water. Reference standards for methionine, serine, and homocysteine were 2-,uI aliquots of 1 M solutions and the standard for thymidylic acid was a 2-~1 aliquot of 0.01 M solution (10 &i). Radioactivity of areas corresponding in mobilities to those of the reference standard was measured using a modification of Bray’s liquid scintillation system (Bray 1960) and a Packard Tri-Carb liquid scintillation spectrometer. Samples with blood from uninfected animals were assayed in parallel determinations and the values were used as corrections for white blood cell and platelet metabolism. In the studies in which compounds were screened as sources
AND CANFIELD
of the methyl groups of methionine and thymidylic acid, radioactivity was reported as analyzed, as cpm/l2-PI aliquot. In studies with compounds which might have effects on methionine and thymidylate biosynthesis, the results were expressed as percentages. RESULTS
The values for radioactivity found as methionine and thymidylic acid in the screen of possible methyl group sources are presented in Table I. The values for [methyZJ4C] choline, 5- [fo~myZ-14C]formyItetrahydrofolic acid, [‘“Cl formaldehyde, L[ring-2-14C] histidine, [methgZJ4C] betaine and [methyl-W] sarcosine were essentially the same for infected and uninfected samples. In contrast, L- [ 3-14C]serine clearly contributed radioactivity in the in vitro biosynthesis of methionine and thymidylic acid by Plasmodium knowlesi. Table II presents results from several experiments in which various compounds were examined for effect on the biosynthesis of methionine and thymidylic acid. In these studies the standard errors of triplicate values from their mean were 7.6 and 9.0% for thymidylic acid and methionine from L- [3-14C] serine, respectively, and 10.0% for methionine from L- [35S] homocysteine. When L- [3-14C] serine was the source of radioactive label, all folic acid derivatives and p-aminobenzoic acid effected small to large increases in radioactivity in thymidylic acid. Methionine synthesis was moderately reduced by all these compounds except tetrahydrofolic acid which had no effect, 5-methyltetrahydrofolic acid which decreased the incorporation to 13c/o, and folic acid which increased the incorporation in one instance. The effects of folic, tetrahydrofolic, and 5-methyltetrahydrofolic acids on the biosynthesis of methionine from L- [ %] homocysteine were slight to moderate increases of radioactivity in methionine.
P,hmodium
knowlesi:
METHIONINE TABLE
Activity in Methionine knowlesi-Infected Experiment
and Thymidylic and Uninfected
AND
THYMIDYLIC
I
Acid following Incubation Erythrocytes with Methyl Methionine
Source
251
ACID
in vitro of Plasmodium Group Sourcesa Thymidylic
acid
Infected 108
112
[methyGW]Choline [methyl-W]Betaine [methyl-W]Sarcosine 5-[form+W]Formyl tetrahydrofolic L-[B-“C]Serine L-[ring-2J4C] histidine [i4C]Formaldehyde L[3-14C]Serine
25 Tj80 636 25
Uninfected
(23-28) (358-833) (546726) (22-26)
27 604 510 24
(24-28) (551-656) (448633) (22-26)
Infected 25 (25) 35 125 25 (22-27)
Uninfected 25 (23-25) 38 117 24 (22-25)
acid 209 (199225)
36 (34-39)
34 (31-39)
30h
41 (3647) 386 (345-397)
58 (55-68) 37 (3440)
a Radioactivity is expressed as cpm/l2 ~1 of hydrolysate parentheses) of determined values from triplicate samples. b Single determination.
Pyrimethamine and 5-fluoroorotic reduced thymidylic acid biosynthesis edly and methionine biosynthesis what.
acid marksome-
DISCUSSION
When it was found that, in addition to thymidylic acid, methionine was biosynthesized in vitro by Plasmodium knowlesi (Smith, McCormick, and Canfield 1976) further study was undertaken to investigate additional methyl group sources and related folate metabolism. In the biosynthesis of methionine, the methyl group which is added to homocysteine may be derived from two possible metabolic pathways. One source may be choline or betaine in the choline-betainemethionine sequence ( Muntz 1950). Data obtained utilizing [vI.&~Z-~C] betaine and [ ~&hyl-~~C] choline as methyl donors failed to demonstrate any radioactive uptake in infected samples when compared to uninfected controls. These results are consistent with and support those of Langer, Phisphumvidhi, Jiamperpoon, and Weidhorn (1969) in P. berghi.
and is presented
324 (316-345) 22 (21-23) 46 (38-53) 319 (316-323) as the average
32 (28-35) 24b 33 (30-36) 31 (27-36) and
range
(in
The other pathway, which was found to be present by Smith, McCormick, and Canfield ( 1976)) produces S-methyltetrahydrofolic acid which is the immediate source of the methyl group in reaction with homocysteine. In its precursor, 5,10-methylenetetrahydrofolic acid which itself is the source of the methyl group of thymidylic acid, the methylene moiety may be derived from sources other than serine. A number of possible sources were investigated. Radioactivity from [ 14C] formaldehyde did not appear in methionine or thymidylic acid, indicating that the reaction of formaldehyde and tetrahydrofolic acid to produce 5,10-methylenetetrahydrofolic acid (Blakely, Ramasastri, and McDougall 1963) did not occur in this system. The methyl group of sarcosine had been identified ‘as a precursor of the 3-carbon of serine in rat liver homogenate (Mackenzie 1955). In the present study although radioactivity from [methyF%] sarcosine appeared in methionine and thymidylic acid, the values of uninfected samples were approximately the same as those of the infected samples; the incorporation is con-
252
SMITH,
MCCORMICK, TABLE
Radioactivity Infected and Compoundsa
in Met,hionine and Thymidylic Uninfected Erythrocytes wit,h
AND
CANFIELD
II
Acid following L-@-W]Serine,
Incubation in Z&O L-CW]Homocysteine,
of Plasmodium knowlesiand Folate-Related
L-[3-%]Serine Thymidylic Experiment Added
number
L-[%]Homocysteine Methionine
Methionine
acid
108
112
116
108
112
116
113
100
100 260 I 30 120 270 290
100 150 160
100
100 70 60 70 60 100
100 50 100
100
compound
p-Aminobenzoic acid Folic acid Diglutamylfolic acid Dihydrofolic acid Tetrahydrofolic acid 5-Methyltetrahydrofolic 5-Fluoroorotic acid Pyrimethamine
140
acid 30
13 13
140
160 170 150 100
60 50
60 90 13
130
120 140 60
a Values are comparisons expressed as: percentages. Radioactivities as cpm/l2-pl aliquot were averaged for triplicate samples, the values of uninfected samples were subtracted from those of infected samples, and the values of samples with added folate-related compounds were then compared to the values of samples with no experiment,al additions. Percentage values greater than 20 were rounded off to the nearest multiple of ten.
sidered to result from metabolic activity other than that of the parasites. The lack of incorporation of radioactivity from L- [ring-2-W] histidine and 5 [ formyltetrahydrofolic acid is consistent with the reported absence of 5,10-methylenetetrahydrofolic acid dehydrogenase in Plasmo&urn Zophurue (Platzer 1972). If present, this enzyme would produce 5,10-methylenetetrahydrofolic acid from 5,10-methenyltetrahydrofolic acid which might be derived from histidine by the formininoglutamic acid pathway (Stokstad and Koch 1967) or from 5-formyltetrahydrofolic acid (Greenberg, Wynston, and Nagabhushanam 1965). These pathways have not been found in malaria parasites. Thus far, serine is the only identified source of the methyl groups of methionine and thymidylic acid biosynthesized in vitro by P. knowlai. The results of‘the studies with pyrimethamine and 5fluoroorotic acid were consistent with their modes of action. Pyrimetha-
mine inhibits dihydrofolic acid reductase (EC 1.5.1.3) Ferone, Burchall, and Hitchings 1969), thus interrupting the metabolic cycle of thymidylic acid synthesis. 5-Fluoroorotic acid may be converted metabolically to S-fluorodeoxyuridylic acid which is a specific inhibitor of thymidylate synthetase (Bosch, Harbers, and Heidelberger 1958). Radioactivity from L- [3-l%] serine appearing in thymidylic acid was reduced by approximately 90% by each of these compounds, as anticipated. At the same time the radioactivity from L- [3-l%] serine and from L- [ %S] homocysteine appearing in methionine was reduced by 5-fluoroerotic acid and pyrimethamine reduced that from L- [3-l%] serine; the reductions are presumed to reflect diminished protein synthesis. Addition of p-aminobenzoic acid, dihydrofolic acid, and tetrahydrofolic acid resulted in marked increases in thymidylic acid derived from L- [3-14C ] serine. There were slight increases with folic acid and
PbS?7IOdiUm
knOWleSi:
METHIONINE
diglutamylfolic acid. Except for folic acid in one instance, none of these compounds induced an increase in the amount of incorporated methionine from L- [ 3-YZ] serine; rather there was no effect or more often a decrease. This is consistent with repression of enzymes in the metabolic system by methionine and its metabolites. In studies with Salmonella typhimurium methionine served as precursor in the synthesis of S-adenosylmethionine and methionyl tRNA. Increased levels of methionine resulted in the repression of activity in both N-5,10-methylenetetrahydrofolic ‘acid reductase and tetrahydropteroylglutamate methyl transferase. This was attributed to methionine by Whitehouse and Smith (1973). However, more recent studies indicate that in mutants having high K, values for S-adenosylmethionine synthetase (EC 2.5.1.6), S-adenosyhnethionine, or a derivative is a corepressor rather than methionine itself ( Hobson 1974). Lawrence (1972) indicated that charged methionyl tRNA works jointly with S-adenosyl methionine in repression. Finally, it was observed in EschericMa coli that supplementation with methionine results in a lowering of activity of cystathionase (EC 4.4.1.8), the enzyme converting cystathionine to homocysteine (Greene, Williams, Kung, Spears, and Weissbach 1973). It is currently believed that folic and diglutamylfolic acid are not utilized per se in malarial folate metabolism (Platzer 1974). The data in Table II indicate that although these compounds were not utilized to the same degree ‘as the other folates or p-aminobenzoic acid in thymidylic acid synthesis, some utilization is indicated by the slight increases in thymidylic acid and decreases in methionine to the same degree as with the other folates. With L- [%] homocysteine, the addition of folic, tetrahydrofolic, and 5methyltetrahydrofolic acids resulted in small to moderate increases in methionine levels when compared to uninfected controls. It is
AND THYMIDYLIC
ACID
253
probable, since these compounds decreased the rate of synthesis when L-[~J~C] serine was utilized as precursor, that the addition of increased levels of homocysteine was adequate to compensate for a lowered activity of cystathionase. The addition of 5methyltetrahydrofolic acid resulted in a further small increase in methionine levels. The reduction of radioactivity in methionine from L- [3-14C] serine to 13% is presumed to be a dilution effect of the exogenous unlabeled 5-methyl group of 5methyltetrahydrofolic acid, or may reflect diminished biosynthesis of 5-methyltetrahydrofolic acid due to inhibition of 5,IOmethylene tetrahydrofolate reductase. The increased levels of thymidylic acid resulting from the addition of 5-methyltetrahydrofolic acid indicate that the methyl group was transferred and that the resulting tetrahydrofolic acid was reutilized by the parasite in the biosynthetic cycle of thymidylic acid. ACKNOWLEDGMENTS The authorsexpress their appreciation to Gloria P. Willet for her contribution to the study, and to Nesbitt D. Brown, of the Division of Biochemistry of this institute, for performance of amino acid analyses. In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care” as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. This is Contribution No. 1432 from the U.S. Army Research Program in Malaria. REFERENCFX BLAKELY, R. L., RAMASASTRI, B. V., AND McDOUGALL, B. M. 1963. The biosynthesis of thymidylic acid. IV. Further studies on thymidylate synthetase. Journal of Biological ChemiStTy 238, 2113-2118. BLAKELY, R. L. 1969. “The Biochemistry of Folic Acid and Related Pteridines,” pp. 231-256, 332-353. John Wiley and Sons, New York. BOSCH, L., HARBERS, E., AND HEIDELBERGER, C. 1958. Studies of fluorinated pyrimidines. V.
254
SMITH,
MCCORMICK,
Effects on nucleic acid metabolism in vitro. Cancer Research 18, 335-343. BRAY, G. S. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analytical Biochemistry 1, 279-285. FERONE, R., BIJRCHALL, J. J., AXD HITCHINGS, G. H. 1969. Plasmodium berghei dihydrofolate reductase. Isolation, properties, and inhibition by antifolates. Molecular Pharmacology 5, 4959. GREENBERG, D. M., WYNSTON, L. K., AND NAGABHUSHANAM, A. 1965. Further studies on N”formyltetrahydrofolic acid cyclodehydrase. Biochemistry 4, 1875-1878. GREENE, R. C., WILLIAMS, R. D., KUNG, H., SPEARS, C., AND WEISSBACH, H. 1973. Effects of methionine and vitamin Blz on the activities of methionine biosynthetic enzymes in Met J Mutants of Escherichia coli K12. Archives of Biochemistry and Biophysics 158, 249-256. HOBSON, A. C. 1974. The regulation of methionine and S-adenosylmethionine biosynthesis and utilization in mutants of Salmonella typhimurium with defects in S-adenosylmethionine synthetase. Molecular and General Genetics 131, 263-273. LANCER, B. W., JR., PHISPHUMVIDHI, P., JIAMPERPOON, D., AND WEIDHORN, R. P. 1969. Malaria parasite metabolism: The metabolism of methionine by Plasmodium berghei. Mi’litary Medicine 10, 1039-1043. LAWRENCE, D. A. 1972. Regulation of the methionine-feedback sensitive enzyme in mutants of Salmonella typhimurium. Journal of Bacteriology 109, 8-11. MACKENZIE, C. G. 1955. Conversion of N-methyl glycines to active formaldehyde and serine. In
AND
CANFIELD
‘Amino Acid Metabolism” ( W. D. McElroy and H. B. Glass, eds.), pp. 684-726. Johns Hopkins Press, Baltimore. MUNTZ, J. A. 1950. The inability of choline to transfer a methyl group directly to homocysteine for methionine formation. Journal of Biological Chemistry 182, 489-499. PLATZER, E. G. 1972. Metabolism of tetrahydrofolate in Plasmodium Zophurae and duckling erythrocytes. Transactions of the New York Academy of Sciences 34, 200-208. PLATZER, E. G. 1974. Dihydrofolate reductase in Plusmodium lophurae and duckling erythrocytes. Journal of Protozoology 21, 400405. REID, V. E., and FRIEDKIN, M. 1973. Thymidylate synthetase in mouse erythrocytes infected with Plasmodium berghei. Molecular Pharmacology 9, 74-80. SOUTH, C. C., MCCORMICK, G. J., AND CANFIELD, C. J. 1976. Plasmodium knowlesi: In vitro biosynthesis of methionine. Experimental Parusitology 40, 432-437. STOKSTAD, E. L. R., AND KOCH, J. 1967. Folic acid metabolism. Physiological Reviews 47, 83-116. WALSH, C. J., AND SHERMAN, I. W. 1968. Purine and pyrimidine synthesis by the avian malaria parasite. Plasm&urn lophurae. Journal of Protozoology 15, 763-770. WALTER, R. D., AND KOXIGK, E. 1971. Synthese der desoxythmyidylat-synthetase und der dihydrofolate-reduktase bei synchroner schizogonie von Plasmodium chabaudi. Zeitschrift fur Tropenmedizin and Parasitologic 22, 250-255. WHITEHOUSE, J. M., AND SMITH, D. A. 1973. Methionine and vitamine Blr repression and precursor induction in the regulation of homocysteine methylation in Salmonella typhimurium. Molecular and General Genetics 120, 341-353.
PARASITOLOGY
Plasmodium
43,
(1977)
knowlesi: In Vitro Biosynthesis and Thymidylic Acid
CRAIG C. SMITH,~ Dicision
248-254
of Medicinal
GEXALD Chemistry, Washington,
(Accepted
for
J, MCCORMICK, Walter D.C. publication
Reed 20012
of Methionine
AND CRAIG J. CANFIELD Army U.S.A.
13 May
Institute
of Research,
1977)
C. C., MCCORMICK, G. J., AND CANFIELD, C. J. 1977. Plas-modium ,&wl.&: biosynthesis of methionine and thymidylic acid. Experimental Parasitology 43, Mcthionine and thymidylic acid biosyntheses were studied to investigate methyl group sources in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with L-[3-‘Clserine, L-[ring-2-l”C]histidine, [“Clformaldehyde, [m&hyGYJcholine, [methyl-‘Clbetaine, [methyL%]sarosine, and 5-[formyE “C] formyltetrahydrofolic acid. Thin layer chromatography of acid hydrolysates of washed erythrocytcs showed that only L-[3-14C]serine was utilized as a methyl donor. In cultures utilizing L-[3-%]serine and L-13”S]homocysteine, the results of studies with pyrimethamine and 5fluoroorotic acid were consistent with their modes of action. An investigation of related folate metabolism revealed that large increases in thymidylic acid synthesis occurred with the addition of p-aminobenzoic, dihydrofolic, and tetrahydrofolic acids, and smaller increases with the addition of folic and diglutamylfolic acids to cultures containing L-[3-“Cl serine. With the exception of tetrahydrofolic acid, which had no apparent effect, all of the previously mentioned compounds induced a decrease in methionine synthesis, attributed to repression by methionine and its metabolic end products. Experiments utilizing L-["S] homocysteine produced small to moderate increases in methionine levels upon addition of folic, tetrahydrofolic, and methyltetrahydrofolic acids, presumably due to increased homocysteine levels, compensating for repressed cystathionase (EC 4.4.1.8) activity. INDEX DESCRIPTORS: Plasmodium knowlesi; Parasitic protozoa; Malaria; Methionine r.-[ring-2-14C]Histidine; [14C]Formaldebiosynthesis; L-[3-%]Serine; L-[YSlHomocysteine; hyde; [methyl-‘“C]Sarcosine; [methyZ-14C]Betaine; [methyZ-14C]Choline; 5-[formyl-‘“C]Formyltetrahydrofolic acid; Tetrahydrofolic acid; Dihydrofolic acid; Folic acid; p-Aminobenzoic acid; Diglutamyl folic acid; S-Methyltetrahydrofolic acid; 5-Fluoroorotic acid; Pyrimethamine; Rhesus monkey; Macacca mdutta; in citro culture; S-Adenosyl methionine; Methionyl tRNA. SMITH,
In vitro 248-254.
The association of folate metabolism and the biosynthesis of methionine and thymidylic acid is well established in mammals and microorganisms (Stokstad and Koch 1967; Blakely 1969). Thymidylic 1 Present address: Section on Adult Psychiatry Branch, National Mental Health, Bethesda, Maryland
Biochemistry, Institute of 20014, U.S.A.
acid is formed by transfer of the methylene group of 5,10-methylenetetrahydrofolic acid to deoxyuridylic acid by the action of thymidylate synthetase (this enzyme has no EC number). After the conversion of 5,10-methylenetetrahydrofolic acid to 5methyltetrahydrofolic acid by 5,10-methylenetetrahydrofolic acid reductase (EC 1.1.1.68), methionine is produced by donation of the methyl group to homo-
218 Copyright All rights
0 1977 by Academic of reproduction in any
Press, Inc. form reserved.
ISSN
0014
4894
Phmodium
knowlesi:
METHIONINE
cysteine by action of tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13) or tetrahydropteroyltriglutamate methyltransferase (EC 2.1.1.4). The initial 5,10methylenetetrahydrofolic acid may be the product of several mechanisms: reduction of 5,10-methenyltetrahydrofolic acid by 5,10-methylenetetrahydrofolic acid dehydrogenase (EC 1.5.1.5), reaction of serine and tetrahydrofolic acid by serine hydroxymethyltransferase (EC 2.1.2.1), and nonenzymatic reaction of formaldehyde and tetrahydrofolic acid (Blakely, Ramasastri and McDougall 1963). There is limited information concerning the presence of these reactions in malaria parasites. Thymidylate synthetase was demonstrated in Plasmodium lophurae by Walsh and Sherman ( RX%), in P. berghei by Reid and Friedkin ( 1973), and in P. chabaudi by Walter and Konigk ( 1971). Platzer (1972) demonstrated the presence of serine hydroxymethyl transferase in P. Zophurae and reported that lo-formyltetrahydrofolate synthetase (EC 6.3.4.3) and 5,10-methylenetetrahydrofolic acid dehydrogenase apparently were absent in this malaria parasite. Evidence of biosynthesis of methionine by P. berghei was obtained by Langer, Phisphumvidi, Jiamperpoon, and Weidhorn ( 1969), and Smith, McCormick, and Canfield (1976) observed biosynthesis of methionine and thymidylic acid by P. knowlesi; the methyl groups were derived from serine. This report describes a study of the association of folates and biosynthesis of methionine and thymidylic acid in P. knowlesi-infected blood in vitro. A survey was made of compounds which are sources of methyl groups in other organisms for possible utilization by this parasite. Evidence of relationship between the biosynthesis of methionine and thymidylic acid and the metabolism of folates was sought by observation of effects of folate and 5intermediates, pyrimethamine, fluoroorotic acid on the appearance in
AND THYMIDYLIC
ACXD
249
methionine and thymidylic acid of radioactivity from L-[3-lkC]serine and L-[““S] homocysteine. MATERIALS
AND METHODS
labeled compounds Radioisotopically used were L-[3-14C]serine (specific activity 56.5 mCi/mmole, L- [YS] homocysteine thiolactone (specific activity 6.1 or 8.2 mCi/ mmole ), and L- [ring-2-14C] histidine ( specific activity 55 mCi/mmole), obtained from AmershamJSearle, [‘Cl formaldehyde ( specific activity 59.2 mCi/mmole), [methyl-W] sarcosine (specific activity 3.86 mCi/mmole, [methy F’C] betaine ( specific activity 2.60 mCi/mmole), and [m&Z‘“Cl choline (specific activity 8.68 mCi/ mmole), obtained from New England Nuclear, and 5- [formyZ-14C]formyltetrahydrofolic acid (folinic acid) (specific activity 2.73 mCi/mmole) obtained from Monsanto Research Corporation. The specific activity of L- [ 3-“C] serine was adjusted to 28.8 mCi/mmole by addition of nonradioactive L-serine. L- [ 731 HOmocysteine thiolactone was supplemented with nonradioactive L-homocysteine, hydrolyzed in 1 N HCI for 24 hr, and neutralized with 1 N NaOH; the final specific activity was 0.37 mCi/mmole. The following quantities of radioactive compounds were added to cultivation tubes: L- [3-lC]serine, 0.7 pmole (20 &i); L-[35S]homocysteine, 54 pmoles (20 &i); L- [ring-2-l%] histidine, 0.18 pmole (10 &i); [14C]formaldehyde, 0.17 mole (10 &i); [methyZ-14C]sarcosine, 2.6 qoles (10 &i); [methyZ-14C] -betaine, 3.8 pmoles ( 10 &i); [ m.ethyZ-14C]choline, 1.15 wales ( 10 &i) ; and 5-[formyZ-14CJformyltetrahydrofolic acid, 1.5 eoles (4.1 &i). When used, tetrahydrofolic acid, dihydrofolic acid, folic acid, diglutamylfolic acid, p-aminobenzoic acid, and 5-fluoroerotic acid were present at a 0.01 mM con5-methyltetrahydrofolic acid centration, was 2.5 x 10m3mM, and pyrimethamine -.
250
SMITH,
MCCORMICK,
was 2 x 1O-5 m&f. All solutions were adjusted to pH 7.6 with 0.1 N NaOH. Procedures employed for cultivation in vitro and analysis were as reported in detail previously (Smith, McCormick, and Canfield 1976). Cultivation was done in Eagle’s minimum essential medium without folic acid, methionine, serine, or homocysteine, but supplemented with fetal bovine serum. The fetal bovine serum contributed 0.145 pmole of serine and 0.038 pmole of methionine per culture tube; homocysteine was not detected. Blood was taken from Plasmodium knowlesi-infected rhesus monkeys (Macacca mulatta) when 20-2570 of the erythrocytes contained a synchronous population of young trophozoites. The blood cells were washed, suspended (15% v/v) in medium, and 0.2ml aliquots were added to cultivation tubes. After incubation for 17 hr, blood cells were packed by centrifugation at 7006 for 15 min at 3-5°C washed three times with cold medium, and hydrolyzed in 6 N WC1 for 24 hr at 90°C. The hydrolysates were evaporated to dryness and soluble products were extracted into final volumes of 0.5 ml of 20% isopropyl alcohol in water. Aliquots (12 PI) were applied to silica gel thin layer chromatography plates and chromatotographic development was accomplished with a 4: 1: 1 mixture of 1-butanol, glacial acetic acid, and distilled water. Reference standards for methionine, serine, and homocysteine were 2-,uI aliquots of 1 M solutions and the standard for thymidylic acid was a 2-~1 aliquot of 0.01 M solution (10 &i). Radioactivity of areas corresponding in mobilities to those of the reference standard was measured using a modification of Bray’s liquid scintillation system (Bray 1960) and a Packard Tri-Carb liquid scintillation spectrometer. Samples with blood from uninfected animals were assayed in parallel determinations and the values were used as corrections for white blood cell and platelet metabolism. In the studies in which compounds were screened as sources
AND CANFIELD
of the methyl groups of methionine and thymidylic acid, radioactivity was reported as analyzed, as cpm/l2-PI aliquot. In studies with compounds which might have effects on methionine and thymidylate biosynthesis, the results were expressed as percentages. RESULTS
The values for radioactivity found as methionine and thymidylic acid in the screen of possible methyl group sources are presented in Table I. The values for [methyZJ4C] choline, 5- [fo~myZ-14C]formyItetrahydrofolic acid, [‘“Cl formaldehyde, L[ring-2-14C] histidine, [methgZJ4C] betaine and [methyl-W] sarcosine were essentially the same for infected and uninfected samples. In contrast, L- [ 3-14C]serine clearly contributed radioactivity in the in vitro biosynthesis of methionine and thymidylic acid by Plasmodium knowlesi. Table II presents results from several experiments in which various compounds were examined for effect on the biosynthesis of methionine and thymidylic acid. In these studies the standard errors of triplicate values from their mean were 7.6 and 9.0% for thymidylic acid and methionine from L- [3-14C] serine, respectively, and 10.0% for methionine from L- [35S] homocysteine. When L- [3-14C] serine was the source of radioactive label, all folic acid derivatives and p-aminobenzoic acid effected small to large increases in radioactivity in thymidylic acid. Methionine synthesis was moderately reduced by all these compounds except tetrahydrofolic acid which had no effect, 5-methyltetrahydrofolic acid which decreased the incorporation to 13c/o, and folic acid which increased the incorporation in one instance. The effects of folic, tetrahydrofolic, and 5-methyltetrahydrofolic acids on the biosynthesis of methionine from L- [ %] homocysteine were slight to moderate increases of radioactivity in methionine.
P,hmodium
knowlesi:
METHIONINE TABLE
Activity in Methionine knowlesi-Infected Experiment
and Thymidylic and Uninfected
AND
THYMIDYLIC
I
Acid following Incubation Erythrocytes with Methyl Methionine
Source
251
ACID
in vitro of Plasmodium Group Sourcesa Thymidylic
acid
Infected 108
112
[methyGW]Choline [methyl-W]Betaine [methyl-W]Sarcosine 5-[form+W]Formyl tetrahydrofolic L-[B-“C]Serine L-[ring-2J4C] histidine [i4C]Formaldehyde L[3-14C]Serine
25 Tj80 636 25
Uninfected
(23-28) (358-833) (546726) (22-26)
27 604 510 24
(24-28) (551-656) (448633) (22-26)
Infected 25 (25) 35 125 25 (22-27)
Uninfected 25 (23-25) 38 117 24 (22-25)
acid 209 (199225)
36 (34-39)
34 (31-39)
30h
41 (3647) 386 (345-397)
58 (55-68) 37 (3440)
a Radioactivity is expressed as cpm/l2 ~1 of hydrolysate parentheses) of determined values from triplicate samples. b Single determination.
Pyrimethamine and 5-fluoroorotic reduced thymidylic acid biosynthesis edly and methionine biosynthesis what.
acid marksome-
DISCUSSION
When it was found that, in addition to thymidylic acid, methionine was biosynthesized in vitro by Plasmodium knowlesi (Smith, McCormick, and Canfield 1976) further study was undertaken to investigate additional methyl group sources and related folate metabolism. In the biosynthesis of methionine, the methyl group which is added to homocysteine may be derived from two possible metabolic pathways. One source may be choline or betaine in the choline-betainemethionine sequence ( Muntz 1950). Data obtained utilizing [vI.&~Z-~C] betaine and [ ~&hyl-~~C] choline as methyl donors failed to demonstrate any radioactive uptake in infected samples when compared to uninfected controls. These results are consistent with and support those of Langer, Phisphumvidhi, Jiamperpoon, and Weidhorn (1969) in P. berghi.
and is presented
324 (316-345) 22 (21-23) 46 (38-53) 319 (316-323) as the average
32 (28-35) 24b 33 (30-36) 31 (27-36) and
range
(in
The other pathway, which was found to be present by Smith, McCormick, and Canfield ( 1976)) produces S-methyltetrahydrofolic acid which is the immediate source of the methyl group in reaction with homocysteine. In its precursor, 5,10-methylenetetrahydrofolic acid which itself is the source of the methyl group of thymidylic acid, the methylene moiety may be derived from sources other than serine. A number of possible sources were investigated. Radioactivity from [ 14C] formaldehyde did not appear in methionine or thymidylic acid, indicating that the reaction of formaldehyde and tetrahydrofolic acid to produce 5,10-methylenetetrahydrofolic acid (Blakely, Ramasastri, and McDougall 1963) did not occur in this system. The methyl group of sarcosine had been identified ‘as a precursor of the 3-carbon of serine in rat liver homogenate (Mackenzie 1955). In the present study although radioactivity from [methyF%] sarcosine appeared in methionine and thymidylic acid, the values of uninfected samples were approximately the same as those of the infected samples; the incorporation is con-
252
SMITH,
MCCORMICK, TABLE
Radioactivity Infected and Compoundsa
in Met,hionine and Thymidylic Uninfected Erythrocytes wit,h
AND
CANFIELD
II
Acid following L-@-W]Serine,
Incubation in Z&O L-CW]Homocysteine,
of Plasmodium knowlesiand Folate-Related
L-[3-%]Serine Thymidylic Experiment Added
number
L-[%]Homocysteine Methionine
Methionine
acid
108
112
116
108
112
116
113
100
100 260 I 30 120 270 290
100 150 160
100
100 70 60 70 60 100
100 50 100
100
compound
p-Aminobenzoic acid Folic acid Diglutamylfolic acid Dihydrofolic acid Tetrahydrofolic acid 5-Methyltetrahydrofolic 5-Fluoroorotic acid Pyrimethamine
140
acid 30
13 13
140
160 170 150 100
60 50
60 90 13
130
120 140 60
a Values are comparisons expressed as: percentages. Radioactivities as cpm/l2-pl aliquot were averaged for triplicate samples, the values of uninfected samples were subtracted from those of infected samples, and the values of samples with added folate-related compounds were then compared to the values of samples with no experiment,al additions. Percentage values greater than 20 were rounded off to the nearest multiple of ten.
sidered to result from metabolic activity other than that of the parasites. The lack of incorporation of radioactivity from L- [ring-2-W] histidine and 5 [ formyltetrahydrofolic acid is consistent with the reported absence of 5,10-methylenetetrahydrofolic acid dehydrogenase in Plasmo&urn Zophurue (Platzer 1972). If present, this enzyme would produce 5,10-methylenetetrahydrofolic acid from 5,10-methenyltetrahydrofolic acid which might be derived from histidine by the formininoglutamic acid pathway (Stokstad and Koch 1967) or from 5-formyltetrahydrofolic acid (Greenberg, Wynston, and Nagabhushanam 1965). These pathways have not been found in malaria parasites. Thus far, serine is the only identified source of the methyl groups of methionine and thymidylic acid biosynthesized in vitro by P. knowlai. The results of‘the studies with pyrimethamine and 5fluoroorotic acid were consistent with their modes of action. Pyrimetha-
mine inhibits dihydrofolic acid reductase (EC 1.5.1.3) Ferone, Burchall, and Hitchings 1969), thus interrupting the metabolic cycle of thymidylic acid synthesis. 5-Fluoroorotic acid may be converted metabolically to S-fluorodeoxyuridylic acid which is a specific inhibitor of thymidylate synthetase (Bosch, Harbers, and Heidelberger 1958). Radioactivity from L- [3-l%] serine appearing in thymidylic acid was reduced by approximately 90% by each of these compounds, as anticipated. At the same time the radioactivity from L- [3-l%] serine and from L- [ %S] homocysteine appearing in methionine was reduced by 5-fluoroerotic acid and pyrimethamine reduced that from L- [3-l%] serine; the reductions are presumed to reflect diminished protein synthesis. Addition of p-aminobenzoic acid, dihydrofolic acid, and tetrahydrofolic acid resulted in marked increases in thymidylic acid derived from L- [3-14C ] serine. There were slight increases with folic acid and
PbS?7IOdiUm
knOWleSi:
METHIONINE
diglutamylfolic acid. Except for folic acid in one instance, none of these compounds induced an increase in the amount of incorporated methionine from L- [ 3-YZ] serine; rather there was no effect or more often a decrease. This is consistent with repression of enzymes in the metabolic system by methionine and its metabolites. In studies with Salmonella typhimurium methionine served as precursor in the synthesis of S-adenosylmethionine and methionyl tRNA. Increased levels of methionine resulted in the repression of activity in both N-5,10-methylenetetrahydrofolic ‘acid reductase and tetrahydropteroylglutamate methyl transferase. This was attributed to methionine by Whitehouse and Smith (1973). However, more recent studies indicate that in mutants having high K, values for S-adenosylmethionine synthetase (EC 2.5.1.6), S-adenosyhnethionine, or a derivative is a corepressor rather than methionine itself ( Hobson 1974). Lawrence (1972) indicated that charged methionyl tRNA works jointly with S-adenosyl methionine in repression. Finally, it was observed in EschericMa coli that supplementation with methionine results in a lowering of activity of cystathionase (EC 4.4.1.8), the enzyme converting cystathionine to homocysteine (Greene, Williams, Kung, Spears, and Weissbach 1973). It is currently believed that folic and diglutamylfolic acid are not utilized per se in malarial folate metabolism (Platzer 1974). The data in Table II indicate that although these compounds were not utilized to the same degree ‘as the other folates or p-aminobenzoic acid in thymidylic acid synthesis, some utilization is indicated by the slight increases in thymidylic acid and decreases in methionine to the same degree as with the other folates. With L- [%] homocysteine, the addition of folic, tetrahydrofolic, and 5methyltetrahydrofolic acids resulted in small to moderate increases in methionine levels when compared to uninfected controls. It is
AND THYMIDYLIC
ACID
253
probable, since these compounds decreased the rate of synthesis when L-[~J~C] serine was utilized as precursor, that the addition of increased levels of homocysteine was adequate to compensate for a lowered activity of cystathionase. The addition of 5methyltetrahydrofolic acid resulted in a further small increase in methionine levels. The reduction of radioactivity in methionine from L- [3-14C] serine to 13% is presumed to be a dilution effect of the exogenous unlabeled 5-methyl group of 5methyltetrahydrofolic acid, or may reflect diminished biosynthesis of 5-methyltetrahydrofolic acid due to inhibition of 5,IOmethylene tetrahydrofolate reductase. The increased levels of thymidylic acid resulting from the addition of 5-methyltetrahydrofolic acid indicate that the methyl group was transferred and that the resulting tetrahydrofolic acid was reutilized by the parasite in the biosynthetic cycle of thymidylic acid. ACKNOWLEDGMENTS The authorsexpress their appreciation to Gloria P. Willet for her contribution to the study, and to Nesbitt D. Brown, of the Division of Biochemistry of this institute, for performance of amino acid analyses. In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care” as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. This is Contribution No. 1432 from the U.S. Army Research Program in Malaria. REFERENCFX BLAKELY, R. L., RAMASASTRI, B. V., AND McDOUGALL, B. M. 1963. The biosynthesis of thymidylic acid. IV. Further studies on thymidylate synthetase. Journal of Biological ChemiStTy 238, 2113-2118. BLAKELY, R. L. 1969. “The Biochemistry of Folic Acid and Related Pteridines,” pp. 231-256, 332-353. John Wiley and Sons, New York. BOSCH, L., HARBERS, E., AND HEIDELBERGER, C. 1958. Studies of fluorinated pyrimidines. V.
254
SMITH,
MCCORMICK,
Effects on nucleic acid metabolism in vitro. Cancer Research 18, 335-343. BRAY, G. S. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analytical Biochemistry 1, 279-285. FERONE, R., BIJRCHALL, J. J., AXD HITCHINGS, G. H. 1969. Plasmodium berghei dihydrofolate reductase. Isolation, properties, and inhibition by antifolates. Molecular Pharmacology 5, 4959. GREENBERG, D. M., WYNSTON, L. K., AND NAGABHUSHANAM, A. 1965. Further studies on N”formyltetrahydrofolic acid cyclodehydrase. Biochemistry 4, 1875-1878. GREENE, R. C., WILLIAMS, R. D., KUNG, H., SPEARS, C., AND WEISSBACH, H. 1973. Effects of methionine and vitamin Blz on the activities of methionine biosynthetic enzymes in Met J Mutants of Escherichia coli K12. Archives of Biochemistry and Biophysics 158, 249-256. HOBSON, A. C. 1974. The regulation of methionine and S-adenosylmethionine biosynthesis and utilization in mutants of Salmonella typhimurium with defects in S-adenosylmethionine synthetase. Molecular and General Genetics 131, 263-273. LANCER, B. W., JR., PHISPHUMVIDHI, P., JIAMPERPOON, D., AND WEIDHORN, R. P. 1969. Malaria parasite metabolism: The metabolism of methionine by Plasmodium berghei. Mi’litary Medicine 10, 1039-1043. LAWRENCE, D. A. 1972. Regulation of the methionine-feedback sensitive enzyme in mutants of Salmonella typhimurium. Journal of Bacteriology 109, 8-11. MACKENZIE, C. G. 1955. Conversion of N-methyl glycines to active formaldehyde and serine. In
AND
CANFIELD
‘Amino Acid Metabolism” ( W. D. McElroy and H. B. Glass, eds.), pp. 684-726. Johns Hopkins Press, Baltimore. MUNTZ, J. A. 1950. The inability of choline to transfer a methyl group directly to homocysteine for methionine formation. Journal of Biological Chemistry 182, 489-499. PLATZER, E. G. 1972. Metabolism of tetrahydrofolate in Plasmodium Zophurae and duckling erythrocytes. Transactions of the New York Academy of Sciences 34, 200-208. PLATZER, E. G. 1974. Dihydrofolate reductase in Plusmodium lophurae and duckling erythrocytes. Journal of Protozoology 21, 400405. REID, V. E., and FRIEDKIN, M. 1973. Thymidylate synthetase in mouse erythrocytes infected with Plasmodium berghei. Molecular Pharmacology 9, 74-80. SOUTH, C. C., MCCORMICK, G. J., AND CANFIELD, C. J. 1976. Plasmodium knowlesi: In vitro biosynthesis of methionine. Experimental Parusitology 40, 432-437. STOKSTAD, E. L. R., AND KOCH, J. 1967. Folic acid metabolism. Physiological Reviews 47, 83-116. WALSH, C. J., AND SHERMAN, I. W. 1968. Purine and pyrimidine synthesis by the avian malaria parasite. Plasm&urn lophurae. Journal of Protozoology 15, 763-770. WALTER, R. D., AND KOXIGK, E. 1971. Synthese der desoxythmyidylat-synthetase und der dihydrofolate-reduktase bei synchroner schizogonie von Plasmodium chabaudi. Zeitschrift fur Tropenmedizin and Parasitologic 22, 250-255. WHITEHOUSE, J. M., AND SMITH, D. A. 1973. Methionine and vitamine Blr repression and precursor induction in the regulation of homocysteine methylation in Salmonella typhimurium. Molecular and General Genetics 120, 341-353.
