Plasmodium lophurae: [U-14C]-glucose catabolism by free plasmodia and duckling host erythrocytes

EXPERIMENTAL

25, 181-192

PARASITOLOGY

Plasmodium

lophorae:

Plasmodia Irwin Department

and

W. Sherman,

( 1969)

[U-‘“Cl

Duckling Judith

of Life Sciences, University (Submitted

-Glucose

by

Free

Host Erythrocytes

A. Ruble,

and

of California,

for publication

Catabolism

Irwin Riverside,

2 October,

P. Ting California

92502

1967)

SHERMAN, IRWIN W., RUBLE, JUDITH A., AND TING, IRWIK P. 1969. Plasmodium lophurae: [U-tJC]-Glucose Catabolism by Free plasmodia and Duckling Host Erythrocytes. Experimental Parasitology 25, 181-192. The catabolism of glucose by the avian malaria parasite Plasmodium Zophurae was studied in vitro by employing the radioisotope [U-14Cl-d-glucose. Quantitatively, infected cells showed more radioisotope in soluble intermediates than did normal duck erythrocytes. Variations in the amount of 14C in these soluble products depended on the medium in which the cells were suspended; normal and Plasmodium-infected erythrocytes had considerably greater amounts of isotope in soluble intermediates when the cells were suspended in plasma rather than in buffered chick embryo Ringer’s solution. P. lophurae had more radioactivity in soluble intermediates than did normal and infected cells in Ringer’s solution and normal cells in plasma; however, the amount of 14C in soluble catabolites in the plasmodium was one third that recovered from infected cells in plasma. Qualitatively, the major end-products of ghrcose metabolism by erythrocyte-free P. lophurae and Plasmodium-infected cells in plasma were lactate and lesser amounts of succinate. The principal amino acids formed from glucose were glutamic acid, and smaller amounts of alanine and aspartic acid. Significant changes in the quality of incorporation of 14C into soluble intermediates and CO, were found when infected and normal cells were suspended in Ringer’s solution rather than in plasma. These results are interpreted to indicate the dominant roles of glycolysis and CO, fixation in the metabolism of this malaria parasite. IXDEX

Phmodium Iophurae; glucose; acid; metabolism; alanine; CO,; glycolysis;

DESCRIPTORS:

acid; aspartic

Glucose was described as a nutritional requirement for the malaria parasite more than half a century ago (Bass and Johns, 1912). Later studies demonstrated that the addition of glucose to suspensions of monkey erythrocytes infected with Plasmodium knowlesi had a beneficial effect on parasite growth, and glucose disappeared rapidly from the medium (Christophers and Fulton, 1938). In addition, the in viva administration of glucose enhanced the parasitemia and prolonged the infection in bird 1 This research was supported by a contract from the Office of Naval Research (Nonr-1842 (07)).

lactate; succinate; malaria.

glutamic

malaria ( Hegner and MacDougall, 1926). Although it has been found that a variety of simple sugars and glycerol is metabolized by malaria parasites (Fulton, 1939; Maier and Coggeshall, 1941) glucose alone probably serves as the principal energy-yielding carbohydrate for these obligate intracellular parasites. Early investigations demonstrated a pronounced accumulation of lactic acid when glucose was metabolized by the malaria parasite (Fulton, 1939; Wendel, 1942; McKee et al., 1946; Ball et al., 1948; Fulton and Spooner, 1956). The enzymic processes involved in glucose degradation have also 181

182

SHERMAN,

RUBLE,

been described and the main pathways appear to be the Embden-Meyerhoff glycolytic scheme and the Krebs’ tricarboxylic acid cycle (Silverman et al., 1944; Speck and Evans, 1945; Bovarnick et al., 1946; Speck et al., 1946; Bowman et al., 1961; Bryant et al., 1964). More recently it has been shown that a variety of malaria parasites is capable of CO2 fixation (Ting and Sherman, 1966; Sherman and Ting, 1966, 1968; Siu, 1967; Nagarajan, 1968); the reactions involved provide a metabolic link with the intermediates of glycolysis and the tricarboxylic acid cycle. To date however, there has been no complete study of the metabolic products of glucose metabolism in any malaria parasite, nor has there been a clarification of the role of glucose in CO, fixation. Particularly lacking are detailed investigations using radioactive tracer techniques which are of great value in identifying derivatives of specific metabolites. The present report illustrates, by the use of radioactive glucose, the kinetics of glucose metabolism in the avian malarial parasite, Plusmodium ~O~~ZIMCZ, in Plascodium-infected erythrocytes and in normal duck erythrocytes, and identifies the principal products formed by these cells. MATERIALS

AND METHODS

Parasitological material. The avian malaria parasite, P. lophurae, was maintained by syringe passage in white Pekin ducklings according to the methods of Trager ( 1950). Blood was withdrawn from the jugular vein into heparinized syringes [l ml of 30 mg heparin/IOO ml 0.85% (w/v) NaCl for every 9 ml of whole blood taken], centrifuged at 15OOg for 10 minutes at 4°C and the buffy coat and plasma removed with a Pasteur pipette. Normal and infected cells were resuspended in normal duck plasma at a concentration of 33% (v/v). In other cases, erythrocytes were washed three times in buffered chick embryo Ringer’s solution

AND TING

(Hale, 1965), and resuspended in the latter at a concentration of 33% (v/v). Erythrocyte-free parasites were prepared according to the methods of Sherman and Hull (1960), except that the treatment with deoxyribonuclease was omitted. Parasites were resuspended in glucose-saline buffer (Sherman and Hull, 1960) at a concentration of 33% (v/v). Smears of erythrocyte and free parasite preparations were made and stained with Giemsa stain; none of the preparations showed contaminating leukocytes or thrombocytes. Glucose utilization. Five-milliliter aliquots of cell suspensions, prepared as described above, were added to 125ml Erlenmeyer flasks. The flasks were specially constructed, and contained a center well to which was added a folded piece of filter KOH. paper saturated with 20% (w/v) Flasks containing the cell suspensions were prewarmed to 38°C for 3 minutes in a rocking water bath, and then each received 5 @Zi of d-glucose-14C (uniformly labeled, specific activity 6.42 mCi/mM, New England Nuclear Corp.) Therefore, each flask contained approximately 12 x lo6 dpm of l’C-glucose; the initial total concentration of glucose in erythrocyte suspensions was 150 mg/lOO ml, and with free parasites 225 mg/lOO ml. The flasks were closed with a rubber vaccine cap. After 15, 30, or 60 minutes of gentle agitation and incubation at 38°C the flasks were taken from the water bath, the vaccine cap removed, and the filter paper withdrawn and placed in scintillation fluid ( Ting and Dugger, 1965). The suspension was centrifuged in the cold (4°C) for 10 minutes at 20,OOOg the supernatant (ca. 3 ml) was removed and distilled water added to the cells to bring the total volume to 5 ml. Supernatant and cells were separately extracted with chloroformmethanol, water and chloroform (Ting and Dugger, 1965). The methanol-water layer was separated from the chloroform layer ( containing lipoidal substances), and the

GLUCOSE

METABOLISM

former further purified by passing it sequentially through a Dowex-50 column (H+ form) and a Dowex-1 column (formate form). Total radioactivity in lipids, organic acids, amino acids, and neutral compounds was determined from duplicate samples (0.2 ml) of the chloroform layer and column eluates. Column eluates were taken to dryness by lyophilization, redissolved in 300 ul of distilled water (amino acids) or 80% (v/v) ethanol (organic acids), and lO-20-ul samples were separated into individual acids by thin-layer chromatography (Ting and Sherman, 1966), Tentative identification of radioactivity in individual compounds was made by preparing autoradiograms of the thinlayer plates using Kodak “no screen” X-ray film. After localization of the radioactive spots the plates were scraped, and the powdered gel transferred to vials containing scintillation fluid. Radioactivity was measured using a Packard liquid scintillation spectrometer with an efficiency of approximately 50%. Purity of the radioactive glucose was checked by one-dimensional paper chromatography (Whatman No. 1) with isopropanol:acetic acid:water (3:l:l). Aniline acid-oxalate reagent and autoradiography were used for identification (Block et al., 1958 ) . RESULTS

Radioactive products of [ U-l*C]-glucose metabolism. Three aspects of glucose metabolism in P. lophurae were studied: 1. A comparison of normal and infected duck erythrocytes in normal duck plasma. 2. A comparison of normal and infected erythrocytes in chick embryo Ringer’s solution. 3. The catabolic products of glucose by erythrocyte-free parasites in glucose-saline buffer. The distributions of radioactivity in glu-

IN

MALARIA

183

case and its catabolites for these three experimental situations are shown in Tables 1, II, and III. A major part of the radioactivity present in soluble metabolic intermediates, derived from 14C-glucose, was recovered in the organic acid fraction when normal and infected red blood cells were suspended in plasma (Table I). In uninfected blood 7090% of the total radioactivity recovered in soluble catabolites was found in the organic acid fraction, and in infected blood approximately 60% of the total was in this same fraction. The organic acids which showed the greatest amount of radioactivity in infected blood were lactate ( >SO%), followed by succinate ( ~20%), then citrate and malate; a similar pattern was seen in normal duck blood. Because red cells and medium were separated and then extracted individually, it was possible to gain some impression of the leakage of these organic acids from the cells into the ‘surrounding medium. Leakage of malate was greatest, somewhat less for lactate, still less for succinate, and least for citrate. These data fit well with the known penetration of these compounds into erythrocytes. The major amino acid,s formed from glucose were glutamate and alanine; aspartate showed the least amount of radioactivity in the amino acid fraction. As one might suspect, based on molecular size and charge, leakage of the dicarboxylic (aspartic and glutamic) acids from the red cells was less than that of the neutral amino acid, alanine. The amount of radioactivity trapped as CO* ‘showed considerable differences between normal and infected blood; e.g., at 60 minutes the l*COz released by infected cells was 20 times greater than that of normal cells. Because the cells were neither killed nor the pH reduced by acid until after the cells were separated from the plasma, it is likely that not all of the COa formed was trapped by the potassium hy-

180 2,132

6,191

19,572

-

30

Normal

310 1,968

267 3,150 6,350 10,248

25,630

-

60

1,900 1,476

5,800 18,lCO 15,400 45,262

45,668

0 20,600 11,400

-

15

cells )

30’

4,200 3,280

11,300 48,660 30,400 102,907

80,851

8,600 0 31,000 20,500

60,000 6,314

12,700 75,720 52,000 151,371

122,325

13,800 0 68,600 17,400

60

TABLE I lophurae-Infectad

Infectedo

and Plasmodium

( dpm/ml

by Normal

2.7 x 10”

490 8,830 2,130 12,560 3.3 x 10”

290 5,000 3,200 9,660

3.0 X 106

570 7,970 5,280 15,710

58,400

2,800

39,000

1,870

60

( dpm/ml

Duck

2,340 41,000 1,870

3O’b

Normal

in Normal

1,570 21,500 1,100

15

Time

Erythrocytes

‘1 Parasitemia = 83%, parasites with 4+ nuclei; - Radioactivity too low for quantitative estimation. b Sample lost for organic acids. (’ These figures do not represent the sum of individual acids, but consist of the counts from the column. d Total glucose present at 0 time = 150 mg/lOO ml.

120 656

1,494

aspartate Glutamate Alanine i\mino acid totalc

Chose” co, Lipids

6,990

-

15’

of [U-I%]-Glucose

Organic acid totalc

Citrate Malate Lactate Succinate

Utilization

81,500

2.6 x 10”

1,230 18,600 30,200 55,900

2.0 X 10G

2,060 22,400 58,000 89,600

313,000

47,000 195,000

8,470 176,000 24,800

60

details)

5,060 117,000 11,300

30’

Infected

(see text for

2.5 X 10s

735 10,900 13,300 27,250

75,700

15,900

4,870 36,400 6,320

15

supernate)

Plasma

5 dLo

F

$

s g fj uz m

GLUCOSE

METABOLISM

droxide in the center well of the reaction flask; these values for CO2 should therefore be regarded more as trends rather than as absolutes. Similarly, striking quantitative differences existed between the soluble intermediates formed by infected and normal cell suspensions in plasma; e.g., the amount of radioactivity in the organic acids from infected cells was 2-6 times greater than that of normal cells, and for amino acids it was S-10 times greater; the radioactivity found in the total soluble intermediates of infected cells was 3-7 times more than that of normal cells. Approximately 75-S0% of the radioactivity was recovered as watersoluble compounds and COZ; the remainder, probably volatile, was lost. Marked differences in glucose catabolism were observed when red blood cells were suspended in buffered chick embryo Ringer’s solution rather than plasma (Table II). Most apparent were the reduced utilization of glucose and the lowered amounts of radioactivity in soluble intermediates. A second feature, perhaps somewhat less obvious, was the nature of the radioactive products. When normal erythrocytes were suspended in Ringer’s solution with 14Cglucose approximately 75% of the total radioactivity in water-soluble intermediates was recovered in the organic acid fraction; a figure comparable to that found for erythrocytes in plasma. However, it was found that the organic acid fraction for infected cells in Ringer’s solution, represented only 1839% of the total radioactivity in soluble intermediates, a pattern distinctly different from that found for infected cells in plasma. This finding was substantiated by a separate experiment in which the only carbohydrate present in the Ringer5 solution was radioactive glucose. The results of this experiment (not shown in the tables) were: organic acids represented 50-76% of the total soluble intermediates in normal erythrocytes, whereas in infected cells these

IN

MALARIA

185

acids represented only 2536%. It is obvious that glucose catabolism by infected cells in Ringer’s solution is less than that found for these cells in plasma, and that there is a suppression of organic acid production; amino acids seem to be formed in similar amounts in both media. The amino acids formed by infected cells in plasma were glutamate > alanine > aspartate whereas in Ringer’s solution glutamate > alanine = aspartate. As indicated above, the values for radioactive COa should be regarded as relative rather than absolute figures. Large differences in amounts of 14C in soluble catabolites and CO, were found when infected and normal blood cells in Ringer’s solution were compared; e.g., the radioactivity in total soluble intermediates in infected cells was 820 times greater than that of normal cells; in the organic acids there was S-7 times more radioactivity in infected blood than in normal blood, and for amino acids there was 8-28 times more isotope in infected blood; 14C02 production by infected blood was 70-440 times that of normal duck blood cells. Plasmodium-infected erythrocytes in Ringer’s solution produced 330 times more lipid from glucose-i4C than did normal cells in the same medium. Erythrocyte-free plasmodia, suspended in glucose-saline buffer, metabolized glucose at about one-third the rate of infected cells in plasma (Table III). Organic acids represented about 80% of the total radioactivity recovered in water-soluble intermediates; a figure comparable to the distribution of radioactivity found for normal cells in plasma or Ringer’s ,solution, and for infected cells in plasma. The major organic acids formed from glucose were lactate, followed by citrate and succinate. The amino acids derived from glucose were principally glutamate with smaller quantities of aspartate and alanine. COB production by free parasites was about the same as that found for infected cells in plasma. Lipid produc-

GlucoseC

Ammo acid tottalb

2,775

1,921

230 492

-

140 1,886

5,592

-

30’

6,524

15’

Normal

[ U-iS]-Glucose

410 328

2,775

-

6,990

60’

15

cells )

10,000 6,560

64,690

9,600 37,550 12,500

36,000 8,364

115,076

10,100 76,450 24,000

44,969

30

180,000 9,512

165,249

25,400 112,900 20,000

15

Time

3.7 x 10s

586

-

3.2 x 10s

731

-

1,424

60’

(dpm/ml

organic

acids.

3.6 x 10s

3,159

360 2,439 259

2,912

15

supernate)

3.4 x 10s

14,600

628 12,786 950

6,116

30

Infected

2.9 x 10”

22,600

588 21,291 746

13,008

60

in Buffered Chick Embryo Ringer’s Solution

3.7 x 10”

731

-

9,184

30

Normal

Erythrocytes

3,344

for details)

30,057

60

text

TABLE II lophurae-Infected (see

Infected”

and Plasmodium

40,076

( dpm/ml

by Normal

a Parasitemia = 85%, parasites with 2-3 nuclei; Radioactivity too low for quanitative evaluation of individual 0 These figures do not represent the sum of individual acids, but consist of the counts. from the column. 0 Total glucose present at 0 time = 150 mg/lOO ml.

co2 Lipids

of

acid totalb

Aspartate Glutamate Alanine

Organic

Utilization

5

5 F P

E “1:

s ii

GLUCOSE METABOLISM

Utilization

TABLE III by Erythrocyte-Free Plasmodium

of [ U-r*C]-Glucose

187

IN MALAIUA

in Glucose-Saline

Iophurae

Solution

(see text for details) Time (dpm/ml 15’ Citrate Malate Lactate Succinate Organic acid totala Aspartate Glutamate Alanine Amino acid total” Glucoseb co, Lipids

cells)

30

(dpm/ml

supernate)

60’

15’

3w

60

4,400 0 11,000 0

4,650 0 7,950 0

5,370 0 29,500 0

710 230 22,000 600

650 2,600 104,000 3,700

900 1,600 87,000 4,300

32,000

59,000

86,000

24,000

110,000

93,000

3,090 13,300 700

2,880 17,500 1,030

5,077 25,400 1,900

-

300 2,800 900

298 3,165 1,140

18,147

22,844

33,946

4,408

4,940

1,200 6,888

10,060 11,398

72,000 19,352

1,824 2.7 x 10s

2.7 x 10s

2.6 x 10s

a These figures do not represent the sum of individual acids, but consist of the counts from the column; - Radioactivity too low for qualitative estimation. b Total glucose present at 0 time = 225 mg/lOO ml. Note: Approximately 30-50% of the radioactivity in organic acids extracted from the cells remained at the origin; this probably represents sugar phosphates.

tion by the parasite was 5-6 times greater than by infected cells in plasma. About 80% of the radioactivity added as 14C-glucose was recovered in water-soluble compounds and COZ; the remainder was lost. DISCUWON

AND CONCLUSION

Carbohydrate metabolism of malarial, plasmodia, particularly the intraerythrocytic stages, has been studied in some detail over the past 30 years (see review of McKee, 1951; Moulder, 1962; Danforth, 1967; Honigberg, 1967). Although our knowledge of the biochemical processes involved in glucose catabolism remains somewhat incomplete, it is nevertheless possible to describe the metabolic pathways in a general way for the various members of the genus Plasmodium. The malaria parasite and the Plasmodium-infected erythrocyte consume considerably more carbohydrate (principally glucose) than does the normal erythrocyte. The principal product of both

aerobic and anaerobic glucose dissimilation is lactate. Under aerobic conditions, some species can oxidize some of the glucose, but this usually represents a small fraction of the total substrate utilized. Our present studies amply con&m the above description, and suggest that the differences in carbohydrate utilization by the species of malarial plasmodia probably involves minor (but nevertheless signticant) changes. The major end-products of glucose dissimilation by erythrocyte-free P. lophurae and lophurae-infected red blood cells in plasma were lactate, and to a lesser extent succinate. These same substances have been described as the major endproducts of carbohydrate metabolism for P. knowlesi (Wendel, 1942; Ball et al., 1948), P. gallinaceum (Speck and Evans, 1945) and P. berghei (Bowman et al., 1960, 1961; Bryant et al., 1964); it should be noted that variations in the amounts of these compounds among these species do exist. Of interest are the amino acids formed from

188

SHERMAN,

RUBLE,

glucose, principally glutamate and to a lesser extent alanine and aspartate. In some cases (e.g., infected cells in plasma) 3540% of the total radioactivity recovered in soluble intermediates is in the amino acid fraction, whereas in other instances (e.g., infected cells in Ringer’s solution) it may represent as much as 80% of the watersoluble intermediates. The production of amino acids from glucose may have some bearing on the results of earlier workers in which only 60-90% of the total glucose carbon could be accounted for by lactate, sucand Evans cinate, and COZ. Moulder (1946) found that amino nitrogen was produced during glucose metabolism in P. gallinaceum. Although the amount of nitrogenous end-products was the same in the presence or absence of glucose, it was found that the major end-product was amino nitrogen in the presence of carbohydrate, but without glucose, ammonia was dominant. Anaerobiosis decreased the amount of amino nitrogen released and prevented the increase in ammonia when glucose was absent. The interpretation of these data has been that in the presence of glucose, proteins are degraded to amino acids, and in the absence of glucose the amino acids are oxidized to ammonia. Since these previous studies on carbohydrate metabolism did not employ isotopic tracers (at that time they were not commonly available) it was impossible to demonstrate unequivocally the source of the amino nitrogen. The present work clearly shows that glucose serves as a precursor for amino acids in P. lophura; presumably the same applies for P. gallinaceum. Bryant et al. (1964) using 14C-glucase have shown that in P. berghei-infected erythrocytes and P. bastianelli-infected red blood cells (Garnham, 1966) amino acids are also derived from glucose. These workers considered that the amino acids were formed via the tricarboxylic acid cycle, but an equally attractive hypothesis is that these acids are formed via CO2 fixation.

AND

TING

Recent studies with P. lophurae, P. knowlesi, and P. berghei show that these plasmodia are capable of fixing CO2 (Sherman and Ting, 1966, 1968; Siu, 1967) and glutamate and aspartate are the principal endproducts of this pathway. Nagarajan (1968) has shown that P. berghei could incorporate 14C02 only in the presence of glucose. Furthermore, these same acids are strikingly increased in the free amino acid pool of Plasmodium-infected erythrocytes ( Sherman and Mudd, 1966). Although amino acids are derived from globin hydrolysis, it is interesting to note that the amino acids found in greatest concentration in the parasite and in the infected cell are the same ones formed via CO2 fixation. In some respects the rapid labeling of amino acids from glucose in PZas-modium-infected cells and in free parasites resembles the pattern seen in brain tissue (Cremer, 1964; Flock et al., 1966; Gaitonde and Richter, 1966; O’Neal and Koeppe, 1966). Bryant et al. (1964) found that free P. berghei showed maximum radioactivity from 14C-glucose in lactate (approximately 80%). In the amino acid fraction, the isotope was principally located in aspartic acid, with lesser amounts in glutamic acid and alanine. These studies on free P. lophurae show a similar pattern for lactate, but in addition some 14C-activity was associated with citrate and succinate. In contrast to the findings on P. berghei, the amino acid with the greatest amount of radioactivity was glutamate and there were lesser amounts in aspartate and alanine. Similar species differences are also found for the Plasmodium-infected cells. Significant changes in both the quantity and the quality of water-soluble intermediates and CO2 were seen when cells were suspended in different media. The total radioactivity recovered in soluble intermediates and CO2 at 66 minutes showed ratios of 4:l:l for infected cells in plasma: infected cells in Ringer’s solution:free para-

GLUCOSE

METABOLISM

sites in glucose-saline. The ratio for normal cells in plasma:Ringer’s solution at 60 minutes was 1O:l. Moreover, in the case of infected cells the organic fraction showed maximum radioactivity when the medium was plasma, but when it was Ringer’s solution maximum radioactivity was in the amino acid fraction. These changes in product formation, effected by changes in the composition of the medium in which infected cells were suspended, were quite unexpected, but the phenomenon is not without precedent. Joshi et al. ( 1962) have shown that in marine plants the pattern of dark incorporation of 14C02 is predominantly (76-93%) into amino acids; in the presence of salt, cell-free extracts of spinach leaves in the dark show a shift in the pattern of labeling from 14C02 to one that resembles that of marine plants. The reduced amount of isotope in soluble intermediates as well as CO2 found in free parasites and erythrocytes in the absence of plasma is probably indicative of the low suitability of glucose-saline and Ringer’s solution. Also, in the case of free parasites the saponin method of liberating parasites may have damaged the capabilities of the parasites for glucose dissimilation. Another possibility is that the free parasites produced volatile components; e.g., acetate, which were not recovered. The mechanism of product formation from glucose by P. Zophurae still remains an area of speculation. A more complete picture of the pathway(s) certainly requires more data, especially studies of the administration of labeled intermediates, determinations of specific activities, and the labeling pattern of these intermediates. With these reservations in mind the experiments presented here do suggest the following: Pyruvate is probably a key intermediate in glucose catabolism. A major fraction of the glucose metabolized by the infected cell and by the plasmodium itself is converted to pyruvate, and then to lactate. A

IN

MALARIA

189

smaller fraction of the pyruvate is probably carboxylated via COz-fixing enzymes to form oxaloacetate, the latter yielding either malate via malate dehydrogenase or aspartate via transamination. Pyruvate may be a precursor of alanine, or alanine may be formed by decarboxylation of aspartate. By entry into the Krebs’ tricarboxylic acid cycle, pyruvate could form succinate, citrate, and cc-ketoglutarate; the latter keto acid by transamination could yield glutamate. Carbon dioxide may be liberated by decarboxylation of the amino acids or by conventional aerobic degradation of glucose. The conclusion drawn from the foregoing and that of previous workers (see Moulder, 1962) is that malaria parasites, in general, possess the same enzymatic machinery for the dissimilation of glucose as the host cell. Why then is the plasmodium an obligate intracellular parasite? The answer probably lies not in the general pattern of carbohydrate metabolism, but rather in the small but significant differences that exist between the host cell and the parasite. Cell-free extracts of P. lophurae and its host erythrocyte do differ qualitatively in a number of glycolytic enzymes; e.g., hexokinase ( Sherman, unpublished), lactate dehydrogenase ( Sherman, 1961)) phosphoglycerate, and pyruvate kinases (Trager, 1967). In addition, at least one enzyme of the tricarboxylic acid cycle differs (malate dehydrogenase, Sherman, 1966) and most of the species lack enzymes of the hexose monophosphate shunt (Fletcher and Maegraith, 1962; Sherman, 1965). P. berghei is an exceptional case (Langer et al., 1967). Although the enzymes of the tricarboxylic acid cycle have been identified in free malaria parasites and in parasitized erythrocytes, the oxidation of pyruvate is low. Of significance is the fact that free parasites produce large quantities of acetate which is not further metabolized, and which is not formed by parasitized erythrocytes

190

SHERMAN,

RUBLE,

(Moulder, 1962). The present isotope studies and those of Bowman et al. (1961) and Bryant et al. (1964) amply support these facts. The basis for the shift in carbohydrate metabolism by the malaria parasite when free of its host cell is not completely understood, but some insight is offered by the studies of Trager (1954, 1966). He found that although the parasite grows intracellularly with pantothenate in the medium, coenzyme A was necessary for extracellular development of the plasmodium. In addition, the parasite was unable to synthesize coenzyme A (Trager, 1954; Bennett and Traeger, 1967), and depended on the synthetic capacity of the host cell for this cofactor. This lack of coenzyme A conveniently explains the reduced rate of pyruvate oxidation, the formation of large amounts of lactate, and the recovery of derivatives of pyruvate formed via CO, fixation. Thus, this metabolic lesion alone may serve to explain much of the carbohydrate metabolism of the malaria parasite, as well as offering an attractive basis for its obligate parasitic nature. REFERENCES BALL, E. G., MCKEE, R. W., ANFINSEN, C. B., CRUZ, W. O., AND GEIMAN, Q. M. 1948. Studies on malarial parasites. IX. Chemical and metabolic changes during growth and multiplication in viuo and in vitro. JOUIWXZof Biological Chemistry 1’75, 547-571. BASS, C. C., AND JOHNS, F. M. 1912. The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. ]ournal of Experimental Medicine 16, 567-

579. BENNETT, T. P., AND TRAGER, W. 1967. Pantothenic acid metabolism during avian malaria infection: Pantothenate kinase activity in duck erythrocytes and in Plasmodium lophurae. Journal of Protozoalogy 14, 214-216. BLOCK, R. J., DIJRRUM, E. L., AND ZWEIG, G. 1958. A Manual of Paper Chromatography and Paper Electrophoresis. 2nd Ed. Academic Press, New York. BOVARNICK, M. R., LINDSAY, A., AND HELLERMAN, L. 1946. Metabolism of the malarial para-

AND TING

site, with reference to the action of antimalarial agents. I. Preparation and properties of Plasmodium Zophume separated from the red cells of duck blood by means of saponin. Journal of Biological Chemistry 163, 523-551. BOWMAN, I. B. R., GRANT, P. T., AND KERMACK, W. 0. 1960. The metabolism of Plasmodium berghei, the malaria parasite of rodents. I. The separation of the erythrocytic form of P. berghei separated from the host cell. Experimental Parasitology 9, 131-136. BOWMAN, I. B. R., GRANT, P. T., KERMACK, W. O., AND OGSTON, D. 1961. The metabolism of PlasmocZium berghei, the malaria parasite of rodents. 2. An effect of mepacrine on the metabolism of glucose by the parasite separated from its host cell. BiochemicaZ Journal 78, 472-478. BHYANT, C., VOLLER, A., AND SMITH, M. J. H. 1964. The incorporation of radioactivity from [14C] glucose into the soluble metabolic intermediates of malaria parasites. American Journal of Tropical Medicine and Hygiene 13, 515-519. CHRISTOPHERS, S. R., AND FULTON, J. D. 1938. Observations on the respiratory metabolism of malaria parasites and trypanosomes. Ann& of Tropical Medicine and Parasitology 32, 4375. CREMER, J. 1964. Amino acid metabolism in rat brain studied with 1%~labelled glucose. Journal of Neurochemistry 11, 165-185. DANFORTH, W. F. 1967. Respiratory metabolism. In “Research in Protozoology” (T. T. Chen, ed.) pp. 201-306. Macmillan (Pergamon), New York. FLETCHER, K. A., AND MAEGRAITH, B. G. 1962. Glucose-B-phosphate and 6-phosphogluconate dehydrogenase activities in erythrocytes of monkeys infected with Plasmodium knowlesi. Nature 196, 1316-1318. FLOCK, E. V., TYCE, G. M., AND OWEN, CHARLES, A., JR. 1966. Utilization of [U-1%] glucose in brain after total hepatectomy in the rat. Journal of Neurochemistry 13, 1389-1406. FULTON, J. D. 1939. Experiments on the utilization of sugars by malaria parasites (PZasmodium knowlesi). Annals of Tropical Medicine and Parasitology 33, 217-227. FULTON, J. D., AND SPOONER, D. F. 1956. The in vitro respiratory metabolism of Plasmodium berghei. Experimental Parasitology 5, 59-78. GAITONDE, M. K., AND RICHTER, D. 1966. Changes with age in the utilization of glucose carbon in liver and brain. Journal of Neurochemistry 13, 1309-1318.

GLUCOSE

METABOLISM

GARNHAM, P. C. C. 1966. “Malaria Parasites and Other Haemosporidia.” Blackwell, Oxford. 1114 pp. HALE, L. J. 1965. “Biological Laboratory Data,” pp. 104-105. Wiley, New York. HEGNER, R. W., AND MAC DOIJCALL, M. S. 1926. Modifying the course of infections with bird malaria by changing the sugar content of the blood. American Journal of Hygiene 6, 602608. HONIGBERG, B. M. 1967. Chemistry of parasitism among some protozoa. In “Chemical Zoology” (G. W. Kidder, ed.) Vol. I. Protozoa. pp. 695-814. Academic Press, New York. Josrrr, G., DOLAN, T., GEE, R., AND SALTMAN, P. 1962. Sodium chloride effect on dark fixation of CO, by marine and terrestrial plants. Plant Physiology 37, 446-449. LANGER, B. W., JR., PHISPH~MVDHI, P., AND F~EDLANDER, Y. 1967. Malarial parasite metaboIism. The pentose cycle in Plasmodium berghei. Experimental Parasitology 20, 68-76. MAIER, J., AND COGGESHALL, L. T. 1941. Respiration of malaria plasmodia. Journal of Infectious Diseases 69, 87-96, MCKEE, R. W. 1951. Biochemistry of Plasmodium and the iniluence of antimalarials. In “Biochemistry and Physiology of Protozoa. ( S. H. Hutner and A. Lwoff, eds.), Vol. 1, pp. 251-322. Academic Press, New York. MCKEE, R. W., ORMSBEE, R. A., ANFINSEN, C. B., GEIMAN, Q. M., AND BALL, E. G. 1946. Studies on malarial parasites. VI. The chemistry and Metabolism of normal and parasitized (P. knowlesi) blood. Journal of Expertmental Medicine 84, 569-621. of IntraMOULDER, J. 1962. “The Biochemistry cellular Parasitism. Pp. 1342. Univ. of Chicago Press, Chicago, Illinois. MOULDER, J. W., AND EVANS, E. A., JR. 1946. The biochemistry of the malaria parasite. VI. Studies on the nitrogen metabolism of the malaria parasite. Journal of Biologicat Chemisty 164, 145-157. NAGARAJAN, K. 1968. Metabolism of Ph~.~modium berghei. III. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids. Experimental Parasitology 22, 3342. O’NEAL, R. M., AND KOEPPE, R. E. 1966. Precursors in uiuo of glutamate, aspartate and their derivatives of rat brain. Journal of Neurochemistry 13, 835-847. SHERMAN, I. W. 1961. Molecular heterogeneity of lactic dehydrogenase in avian malaria (Plasmodium lophurae) . Journal of Experimental Medicine 114, 1049-1062.

IN

MALARIA

191

SHERMAN, I. W. 1965. Glucose+phosphate dehydrogenase and reduced glutathione in malaria-infected erythrocytes (Plasmodium lophume and P. berghei. ) Journal of Protozoology 12, 394-396. SHERMAN, I. W. 1966. Malic dehydrogenase heterogeneity in malaria (Plasmodium Zophurae and P. berghei). Journal of Protozoology 12, 394-396. SHERMAN, I. W., AND HULL, R. W. 1960. The pigment (hemozoin) and proteins of the avian malaria parasite Plasmodium lophurae. Journal of Protozoology 7, 409416. SHERMAN, I. W., AND M~DD, J. B. 1966. Malaria infection (Phzsmodium Zophurae) : Changes in free amino acids. Science 154, 287-289. SHERMAN, I. W., AND TING, I. P. 1966. Carbon dioxide fixation in malaria (Plasmodium lophurue). Nature 212, 1387-1389. SHERMAN, I. W., AND TINT,, I. P. 1968. Carbon dioxide fixation in malaria. II. Plasmodium knowlesi (monkey malaria). Comparative Biochemistry and Physiology 24, 639-642. SILVERMAN, M., CEITHAML, J., TALIAFERRO, L. G., AND EVANS, E. A., JR. 1944. The in vitro metabolism of Plasmodium gallinaceum. Journal of Infectious Diseases 75, 212-230. SIU, P. M. L. 1967. Carbon dioxide fixation in plasmodia and the effect of some antimalarial drugs on the enzyme. Comparative Biochemistry and Physiology 23, 785-795. SPECK, J. F., AND EVANS, E. A., JR. 1945. The biochemistry of the malaria parasite. II. Glycolysis in cell-free preparations of the malaria parasite. Journal of Biological Chemistry 159, 71-81. SPECK, J. F., MOULDER, J. W., AND EVANS, E. A., JR. 1946. The biochemistry of the malaria parasite. V. Mechanism of pyruvate oxidation in the malaria parasite. Journal of biological Chemistry 164, 119-144. TING, I. P., AND DUGGER, W. M., JR. 1965. Separation and detection of organic acids on silica gel. AnaZytical Biochemistry 12, 571578. TING, I. P., AND SHERMAN, I. W. 1966. Carbon dioxide fixation in malaria, I. Kinetic studies in Plasmodium lophurae. Comparative Biochemistry and Ph!ysioZogy 19, 855-869. TRAGER, W. 1950. Studies on the extracellular cultivation of intracellular parasite (avian malaria). I. Development of the organisms in erythrocyte extracts and the favoring effect of adenosine triphosphate. Journal of Erper+ mental Medicine 74, 441461.

192

SHERMAN,

RUBLE,

W. 1954. Coenzyme A and the malaria parasite P&no&urn lophurae. Joumzal of Protozoology 1, 231-237. TRAGER, W. 1966. Coenzyme A and the antimalarial action in vitro of antipantothenate against Plasmodium lophurae, P. coatneyi, and P. falciparum. Transactions of the New York Academy of Sciences Series II 28, 1094-1101.

TRAGER,

AND

TING

W. 1967. Adenosine triphosphate and the pyruvic and phosphoglyceric kinases of the malaria parasite Plasmodium Zophurae. Journal of Protozoology 14, 110-114. WENDEL, W. B. 1942. Respiratory and carbohydrate metabolism of malaria parasites (PZusknow&). JoumaZ of Biological modium Chemisty 148, 21-34. TRAGER,