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Malarial parasite metabolism: The lactic acid dehydrogenase of Plasmodium berghei
EXPERIMENTAL
PARASITOLOGY
Malarial
Parasite
Pirom Department
24, 37-41
(1969)
Metabolism: The Lactic Acid of Plasmodium berghei’ Phisphumvidhi
of Parasitology,
Medical
(Submitted
and
Bernhardt
Research Project, for publication,
W.
Langer,
Rajavithi
Dehydrogenase
Jr.?
Road, Bangkok,
Thailand
2 July 1966)
PHISPHUMVIDHI, P., AND LANGER, B. 1969. Malarial Parasite Metabolism: The Lactic Acid Dehydrogenase of Plasmodium berghei. Experimental Parasitology 24, 3741. The electrophoretic mobility, substrate stereospecificity, and apparent Michaelis constant (K,) values for lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized and reduced forms) were examined for the lactic dehydrogenase (LDH) of cell-free extracts of normal mouse erythrocytes and host cell-free P. berghei. The parasite enzyme was electrophoretically distinct from the LDH of the host cell. Both LDH’s were stereospecific for L( +) lactic acid. No significant differences existed between the host cell and the parasitic LDH’s in the K, values for lactic acid and nicotinamide adenine dinucleoticle (oxidized and reduced forms). However, the value for pyruvate with the parasite LDH was 1/36th that of the host cell enzyme showing an increased affinity of the former system for pyruvate. The parasitic LDH had higher specific acitivities with both lactic acid and pyruvic acid than did the host cell enzyme. INDEX DESCRIPTORS: Plasmodium berghei; Plasmodium gallinaceum; Plasmodium Zophurae; enzymes rodent malaria; enzymes mouse erythrocyte; electrophoresis; Michaelis constants: enzymes avian malaria; lactic dehydrogenase; metabolism malarial; lactic dehydrogenase; malaria.
This report presents the results of investigations on the electrophoretic mobility, substrate stereospecificity, and K, values of lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized ( NAD) and reduced (NADH) forms) for the LDH’s (Llactate: NAD oxidoreductase; EC 1.1.1.27) of Plasmodium berghei and normal mouse erythrocytes.
Speck and Evans ( 1945), using cell-free extracts of Plasmodium gallinaceum, were the first to demonstrate the presence of the enzyme, lactic acid dehydrogenase ( LDH ) in a malaria parasite. Sherman (1961, 1962) has shown that the LDH of P. Zophurae and that of P. berghei are electrophoretically distinct from that of the host cell. In his studies on the LDH of P. Zophurae and that of the duck erythrocyte, Sherman (1961) determined the Michaelis constant ( Km ) for pyruvate (1.9 X lo-” M for the parasite enzyme and 1.7 x 106” M for the erythrocyte enzyme) and the pH optimum (pH 7.5 for both systems). 1 This is contribution Army Research Program
number 373 from in Malaria.
MATERIALS
AND
METHODS
Chemicals. All chemicals used in this study were of the highest purity commercially available and were utilized without further purification. The amount of L( + ) lactic acid contamination of D( -) isomer was determined using a commercially available test kit for L( + ) lactic acid (Calbiochem, Los Angeles, California 90054).
the
Project, s Alternate address: SEATO Medical U.S. Component, APO San Francisco, 96346 37
38
PHISPHUMVIDHI
Normal and infected blood. Normal or infected blood was collected in a heparinized capillary pipette from the cut aorta of anesthetized mice. P. berghei (NYU-2 mouse strain) infection was induced by intraperitoneal injection of 0.1 ml of infected mouse blood, 5-7 days prior to sacrifice. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed. Isolation of leukocyte-free erythrocytes. Blood from normal mice, collected as described above, was placed in cellulose nitrate centrifuge tubes and centrifuged at 1OOOgfor 20 minutes followed by 10 minutes at 4000g at 4°C. The plasma was carefully removed and the bottom of the centrifuge tube punctured with a double-ended 1%gauge needle. The erythrocytes were removed through this needle by applying a light, but constant, pressure to the top of the centrifuge tube by means of a syringe connected to the centrifuge tube by a rubber stopper. It was found that if only about 757* of the erythrocytes were removed bv this method, no leukocytes could be detected upon microscopic examination. The isolation of P. berghei from the host erythrocyte, preparation of cell-free homogenates, and the determination of hemoglo-
AND
LANGER
bin, total protein, and active protein have been described previously (Langer et al., 1967). LDH determination with lactate as substrate. The LDH activity, with lactate as substrate, was determined using a modification of the method described by Hohorst (1963). Two and one-half milliliters of glytine-hydrazine buffer (0.5 M glycine buffer, pH 9.0; 0.4 M hydrazine) were placed in a quartz spectrophotometer cuvette. Twotenths milliliter of NAD (concentration dependent upon experiment) was added followed by 0.01 ml of erythrocyte extract and 0.09 ml of Tris-buffered Ringers solution (RT) (for preparation, see Langer et al., lot. cit.) or 0.1 ml of the P. berghei extract. To the experimental cuvette was added 0.1 ml of lactate (isomer and concentration used depended upon the experiment) while 0.1 ml of RT was added to the control cuvette. The increase in absorbance (A) at 340 my was followed in a spectrophotometer with a controlled-temperature sample compartment set at 37°C for a period of 5 minutes with readings taken every minute. The specific activity, pmoles of NAD reduced per milligram active protein per minute, was calculated using the formula :
(A&o mr) (2.9) (tmill) (6.2) (ml of cell extract/cuvette) where AA = A, = 5 mill - A, = 0 Inin, 2.9 is the total reaction volume, tlllin is 5, and 6.2 is the umolar extinction coefficient of NADH at 340 mp. LDH determination with pyruvate as substrate. The determination of LDH activity with pyruvate as substrate was conducted using a modification of the method described by Bergmeyer et al. (1963). Into a quartz spectrophotometer cuvette was placed 2.695 ml of R-T, 0.1 ml NADH (concentration dependent upon experiment ) and 0.005 ml of either erythrocyte or P.
(mg active protein/ml
cell extract)
berghei extract. One-tenth milliliter of pyruvate (concentration dependent upon experiment) was added to the experimental cuvette while 0.1 ml R-T was added to the control cuvette. The decrease in absorbance (A) at 366 mu was followed in a spectrophotometer with a controlled-temperature sample compartment set at 37°C for a period of 5 minutes with readings being taken every minute. The specific activity, [*moles of NADH oxidized per milligram active protein per minute, was calculated using the formula:
MALARIAL
39
PARASITE METABOLISM
(tmill) (3.3) (ml of cell extract/cuvette) where AA = Ar = Clmi,,- Ar = ;, ,,,in, 2.9 is the total reaction volume, tmill is 5, and 3.3 is the pmolar extinction coefficient of NADH at 366 mu. Electrophoresis. The electrophoretic studies on the P. berghei and erythrocytic LDH’s were conducted on polyacrylamide gel using a modification of the method used by Kirkman (Kirkman, personal communication) for the study of glucose-Sphosphate dehydrogenase. The gel was prepared as follows: 1 part of the mixture of 1 N HCl, 48 ml; Tris, 5.98 gm; Temed, 0.46 ml; HZ0 to 100 ml ( pH 6.66.8), 2 parts of the mixture of acrylamide, 10 gm; Bis, 2.5 gm, H-0 to 100 ml, 1 part of 4 mg riboflavin in 100 ml of HZO, 4 parts of 60% sucrose, and 0.0002 gm of NAD. The mixture was polymerized in the electrophoresis tubes for 30 minutes. The anodic (lower ) and cathodic (upper) buffers were the same (Tris, 14.4 gm; glycine, 3 gm; water to 1 liter ) except that the cathodic buffer was 1 x 10-j M in NAD and contained 2 ml of tracking dye per liter of buffer. Entrapped air was displaced from the top of the electrophoresis column with cathodic buffer and 0.01 ml of the sample (1 vol extract to 1 vol 10% sucrose) was gently layered on top of the gel. Current was applied for approximately 30 minutes using care to maintain the amperage between 4-5 mA. After the electrophoretic run was complete the gels were removed from the tubes and, incubated for 15-30 minutes, in the dark, at 37°C in a reaction mixture consisting of 1 ml of phosphate buffer (0.1 M, pH 7.5), 0.05 ml NaCN (0.1 M). 0.2 ml L( +) lactic acid ( 1.0 M ), 0.2 ml NAD ( 10 mg/ml), 0.05 ml phenazine methosulfate ( 1 mg/ml), 0.5 ml p-iodonitrotetrazolium violet (1 mg/ ml), and 2 ml of 0.2% gelatin.
Michuelis constant (K,,,) determination. The K,,, values for lactic acid, pyruvic acid,
(mg active protein/ml
cell extract)
NAD, and NADH were determined using at least a loo-fold concentration range of the variable under studv. The K,, values were calculated using the reciprocal plot method of Lineweaver and Burk (1934). RESULTS ASD DISCUSSION
All concentrations shown in the figures and tables are the final concentrations of the compound in the reaction mixture. The results are the average of at least two separate trials. It has been established that P. berghei and P. Zophurae have LDH’s that are electrophoretically distinct from that found in the host erythrocyte (Sherman 1961, 1962). Using polyacrylamide gel (disc) electrophoresis we have confirmed the fact that P. berghei LDH is a distinct electrophoretic entity (Fig. 1). However, in contrast to the work of Sherman (1961, 1962) it was observed that the parasite enzyme could not be detected in the erythrocyte of the infected animal. The LDH of the normal mouse erythrocyte was found to separate into four distinct isozymes but no attempt was made to compare the relative activities of the isozymes on a quantitative basis. The LDH of P. berghei was found to be stereospecific for the L( + ) isomer of lactic acid. The D ( - ) isomer showed no activity when corrections were made for the 3.3% L ( + ) isomer contamination. Similar results were observed with mouse ervthrocvte LDH. While the apparent K,, values for lactic acid, NAD, or NADH showed no major differences, the parasite LDH had a K, value for pyruvate that was 1/36th that of the host cell enzyme (Table I). This indicates that the parasite enzyme has a greater affinity for pyruvate than does the host cell enzvme. P. berghi LDH also eshibited
40
PHISPHtJhiVIDHI
AND
LANGER
ORIGIN
/
J 0 tracings resulting from the scan of polyacrylamide electrophoretic gels stained FIG. 1. Densitometer specifically for LDH. (A = extract of normal mouse erythrocytes; B = extract of infected mouse erythrocytes; C = extract of host cell-free I’. herghei. )
TABLE I Michaelis Constants (K,) for the LDH in CellFree Extracts of Host Cell-Free P. berghei and Normal Mouse Erythrocyte@ K, Substrate or cofactor tested L( +)
acid” Pyruvic NADd NADH”
TABLE II Activity of the LDH in Cell-Free Extracts of host Cell-Free P. berghei and Normal Mouse Eythrocytesa Specific activity of the LDH of
values for the LDH of
P. berghei
Normal mouse erythrocvtes Substrate
Lactic acidr
Specific
1.1 5.8 3.1 2.0
x x x x
lo-2M lo-sM lo-4M lo-4M
1.4 1.6 5.9 1.3
x x x x
lOWeM lo-“M lo-“M lo-4M
a The values were determined as described in Materials and Methods. b L( +) lactic acid concentration range tested, 3.4 x 10-a-3.4 x lo--‘M; NAD concentration, 3.7 x lo-“M. c Pyruvic acid concentration range tested, 1.7 x 10-a-1.7 x lo-GM; NADH concentration, 6.9 x lo-4M. * NAD concentration range tested, 3.7 X lo-s3.7 x lo-5M; L( +) lactic acid concentration, 1.7 x lOWaM. (’ NADH concentration range tested, 6.9 x 10-4-1.7 x IO-sM; pyruvic acid concentration, 1.7 x lo-“M.
higher specific activities for the substrates tested than did the host cell LDH; specifically, 6 times greater for lactic acid and 15 times greater for pyruvic acid ( Table II ) . It is interesting to note that the apparent K, value for pyruvate of the LDH of P.
L( + ) Lactic Pyruvic acid
acid
P. berghei
Normal mouse erythrocytes
0.38” 2.8~
0.06” 0.18’
a The values were determined as described in Materials and Methods. b Fmoles NAD reduced/mg. active protein/minute; L( +) lactic acid concentration, 1.7 x lo-2M; NAD concentration, 3.7 X lo-:iM. L’ kmoles NADH oxidized/mg active protein/ min; pyruvic acid concentration, 1.7 x lo-aM; NADH concentration, 6.9 x lo--‘M.
Zophurue (1.9 x lo-” M) Sherman 1961) is of the same order of magnitude as was the value determined for P. berghei (5.8 x lo-” M) . Duck erythrocytic LDH has a K, value for pyruvate of 1.7 x 106” M, not significantly different from that observed for the P. Zophurae LDH (Sherman 1961) thereby showing species differences in the host-parasite relationship when compared to the data presented in this communication for P. berghei and the mouse erythrocyte. Sherman (1961) also noted that P. lophurae LDH was 2.7 times as active as the duck
MALARIAL
PARASITE
erythrocytic LDH with pyruvic acid as substrate, further pointing out species differences in the host-parasite relationship. In view of the fact that the affinity for pyruvate and the specific activity with pyruvate as substrate was greater for the parasite enzyme than it was for the host cell enzyme, the conclusion that the parasite could effectively remove pyruvate from the host cell for its own use, seems logical. Since pyruvate and lactate are the end products of glycdytic carbohydrate metabolism and it is generally conceded that the erythrocyte does not contain an active citric acid cycle, it appears that the parasite would not interfere with erythrocyte metabolism by removing erythrocytic pyruvate. However, because of the accumulation of lactate resulting from the parasite metabolism of its own and “stolen” pyruvate, a decrease in erythrocytic intracellular pH would be expected, assuming the lactate to be ready diffusible across the parasite cell membrane. The specific effects of this drop in pH on the life span and metabolism of the host erythrocyte and on the in viva development of the parasite is unknown. However, Trager (1953, 1957) noted that the cultivation of P. lophurae was most successful at pH 6.8-6.9. Whether the same can be said about the development of P. berghei has not been investigated. It seems logical to postulate that the higher affinity and activity of parasite LDH for pyruvate results in the accumulation of excess lactate that may be necessary for the maintenance of a slightly acid pH required for the development of the malaria parasite.
41
METABOLISM
ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mrs. Duangduen Jiampermpoon and Mr. Mood Saengsri. REFEHENCES BERGMEYER, H. U., BERNT, E., AND HESS, B. 1963. Lactic dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed. ), pp. 736-741. Academic Press, New York. HOHORST, H. J. 1963. L( +) lactate. Determination with lactic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer. ed. ), pp. 266-270. Academic Press, New York. LANCER, B. W., JR., PHISPHUMVIDHI, P., AXD FRIEDLANDER, Y. 1967. Malarial parasite metabolism: The pentose cycle in Plasmodium berghei. Experimental Parasitology 20, 68-76. LINEWEAVER, H. AND BURK, D. 1934. The determination of enzyme dissociation constants. Journal of the American Chemical Society 56, 658-666. SHERMAN, I. W. 1961. Molecular heterogeneity of lactic dehydrogenase in avian malaria (Plasmodium lophurae) . Journal of Experimental Medicine 114, 1049-1062. SHERMAN, I. W. 1962. Heterogeneity of lactic dehydrogenase in intraerythrocytic parasites. Transactions of the New York Academy of Sciences Ser. II, 24, 944-953. 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. TRACER, W. 1953. Further studies on the extracellular cultivation of an avian malaria parasite. Annals of the Nezc; York Academy of Sciences 56, 1074-1093. TRAGER, W. 1957. The nutrition of an intracellular parasite (avian malaria). Acta Tropica 14, 289-301.
PARASITOLOGY
Malarial
Parasite
Pirom Department
24, 37-41
(1969)
Metabolism: The Lactic Acid of Plasmodium berghei’ Phisphumvidhi
of Parasitology,
Medical
(Submitted
and
Bernhardt
Research Project, for publication,
W.
Langer,
Rajavithi
Dehydrogenase
Jr.?
Road, Bangkok,
Thailand
2 July 1966)
PHISPHUMVIDHI, P., AND LANGER, B. 1969. Malarial Parasite Metabolism: The Lactic Acid Dehydrogenase of Plasmodium berghei. Experimental Parasitology 24, 3741. The electrophoretic mobility, substrate stereospecificity, and apparent Michaelis constant (K,) values for lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized and reduced forms) were examined for the lactic dehydrogenase (LDH) of cell-free extracts of normal mouse erythrocytes and host cell-free P. berghei. The parasite enzyme was electrophoretically distinct from the LDH of the host cell. Both LDH’s were stereospecific for L( +) lactic acid. No significant differences existed between the host cell and the parasitic LDH’s in the K, values for lactic acid and nicotinamide adenine dinucleoticle (oxidized and reduced forms). However, the value for pyruvate with the parasite LDH was 1/36th that of the host cell enzyme showing an increased affinity of the former system for pyruvate. The parasitic LDH had higher specific acitivities with both lactic acid and pyruvic acid than did the host cell enzyme. INDEX DESCRIPTORS: Plasmodium berghei; Plasmodium gallinaceum; Plasmodium Zophurae; enzymes rodent malaria; enzymes mouse erythrocyte; electrophoresis; Michaelis constants: enzymes avian malaria; lactic dehydrogenase; metabolism malarial; lactic dehydrogenase; malaria.
This report presents the results of investigations on the electrophoretic mobility, substrate stereospecificity, and K, values of lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized ( NAD) and reduced (NADH) forms) for the LDH’s (Llactate: NAD oxidoreductase; EC 1.1.1.27) of Plasmodium berghei and normal mouse erythrocytes.
Speck and Evans ( 1945), using cell-free extracts of Plasmodium gallinaceum, were the first to demonstrate the presence of the enzyme, lactic acid dehydrogenase ( LDH ) in a malaria parasite. Sherman (1961, 1962) has shown that the LDH of P. Zophurae and that of P. berghei are electrophoretically distinct from that of the host cell. In his studies on the LDH of P. Zophurae and that of the duck erythrocyte, Sherman (1961) determined the Michaelis constant ( Km ) for pyruvate (1.9 X lo-” M for the parasite enzyme and 1.7 x 106” M for the erythrocyte enzyme) and the pH optimum (pH 7.5 for both systems). 1 This is contribution Army Research Program
number 373 from in Malaria.
MATERIALS
AND
METHODS
Chemicals. All chemicals used in this study were of the highest purity commercially available and were utilized without further purification. The amount of L( + ) lactic acid contamination of D( -) isomer was determined using a commercially available test kit for L( + ) lactic acid (Calbiochem, Los Angeles, California 90054).
the
Project, s Alternate address: SEATO Medical U.S. Component, APO San Francisco, 96346 37
38
PHISPHUMVIDHI
Normal and infected blood. Normal or infected blood was collected in a heparinized capillary pipette from the cut aorta of anesthetized mice. P. berghei (NYU-2 mouse strain) infection was induced by intraperitoneal injection of 0.1 ml of infected mouse blood, 5-7 days prior to sacrifice. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed. Isolation of leukocyte-free erythrocytes. Blood from normal mice, collected as described above, was placed in cellulose nitrate centrifuge tubes and centrifuged at 1OOOgfor 20 minutes followed by 10 minutes at 4000g at 4°C. The plasma was carefully removed and the bottom of the centrifuge tube punctured with a double-ended 1%gauge needle. The erythrocytes were removed through this needle by applying a light, but constant, pressure to the top of the centrifuge tube by means of a syringe connected to the centrifuge tube by a rubber stopper. It was found that if only about 757* of the erythrocytes were removed bv this method, no leukocytes could be detected upon microscopic examination. The isolation of P. berghei from the host erythrocyte, preparation of cell-free homogenates, and the determination of hemoglo-
AND
LANGER
bin, total protein, and active protein have been described previously (Langer et al., 1967). LDH determination with lactate as substrate. The LDH activity, with lactate as substrate, was determined using a modification of the method described by Hohorst (1963). Two and one-half milliliters of glytine-hydrazine buffer (0.5 M glycine buffer, pH 9.0; 0.4 M hydrazine) were placed in a quartz spectrophotometer cuvette. Twotenths milliliter of NAD (concentration dependent upon experiment) was added followed by 0.01 ml of erythrocyte extract and 0.09 ml of Tris-buffered Ringers solution (RT) (for preparation, see Langer et al., lot. cit.) or 0.1 ml of the P. berghei extract. To the experimental cuvette was added 0.1 ml of lactate (isomer and concentration used depended upon the experiment) while 0.1 ml of RT was added to the control cuvette. The increase in absorbance (A) at 340 my was followed in a spectrophotometer with a controlled-temperature sample compartment set at 37°C for a period of 5 minutes with readings taken every minute. The specific activity, pmoles of NAD reduced per milligram active protein per minute, was calculated using the formula :
(A&o mr) (2.9) (tmill) (6.2) (ml of cell extract/cuvette) where AA = A, = 5 mill - A, = 0 Inin, 2.9 is the total reaction volume, tlllin is 5, and 6.2 is the umolar extinction coefficient of NADH at 340 mp. LDH determination with pyruvate as substrate. The determination of LDH activity with pyruvate as substrate was conducted using a modification of the method described by Bergmeyer et al. (1963). Into a quartz spectrophotometer cuvette was placed 2.695 ml of R-T, 0.1 ml NADH (concentration dependent upon experiment ) and 0.005 ml of either erythrocyte or P.
(mg active protein/ml
cell extract)
berghei extract. One-tenth milliliter of pyruvate (concentration dependent upon experiment) was added to the experimental cuvette while 0.1 ml R-T was added to the control cuvette. The decrease in absorbance (A) at 366 mu was followed in a spectrophotometer with a controlled-temperature sample compartment set at 37°C for a period of 5 minutes with readings being taken every minute. The specific activity, [*moles of NADH oxidized per milligram active protein per minute, was calculated using the formula:
MALARIAL
39
PARASITE METABOLISM
(tmill) (3.3) (ml of cell extract/cuvette) where AA = Ar = Clmi,,- Ar = ;, ,,,in, 2.9 is the total reaction volume, tmill is 5, and 3.3 is the pmolar extinction coefficient of NADH at 366 mu. Electrophoresis. The electrophoretic studies on the P. berghei and erythrocytic LDH’s were conducted on polyacrylamide gel using a modification of the method used by Kirkman (Kirkman, personal communication) for the study of glucose-Sphosphate dehydrogenase. The gel was prepared as follows: 1 part of the mixture of 1 N HCl, 48 ml; Tris, 5.98 gm; Temed, 0.46 ml; HZ0 to 100 ml ( pH 6.66.8), 2 parts of the mixture of acrylamide, 10 gm; Bis, 2.5 gm, H-0 to 100 ml, 1 part of 4 mg riboflavin in 100 ml of HZO, 4 parts of 60% sucrose, and 0.0002 gm of NAD. The mixture was polymerized in the electrophoresis tubes for 30 minutes. The anodic (lower ) and cathodic (upper) buffers were the same (Tris, 14.4 gm; glycine, 3 gm; water to 1 liter ) except that the cathodic buffer was 1 x 10-j M in NAD and contained 2 ml of tracking dye per liter of buffer. Entrapped air was displaced from the top of the electrophoresis column with cathodic buffer and 0.01 ml of the sample (1 vol extract to 1 vol 10% sucrose) was gently layered on top of the gel. Current was applied for approximately 30 minutes using care to maintain the amperage between 4-5 mA. After the electrophoretic run was complete the gels were removed from the tubes and, incubated for 15-30 minutes, in the dark, at 37°C in a reaction mixture consisting of 1 ml of phosphate buffer (0.1 M, pH 7.5), 0.05 ml NaCN (0.1 M). 0.2 ml L( +) lactic acid ( 1.0 M ), 0.2 ml NAD ( 10 mg/ml), 0.05 ml phenazine methosulfate ( 1 mg/ml), 0.5 ml p-iodonitrotetrazolium violet (1 mg/ ml), and 2 ml of 0.2% gelatin.
Michuelis constant (K,,,) determination. The K,,, values for lactic acid, pyruvic acid,
(mg active protein/ml
cell extract)
NAD, and NADH were determined using at least a loo-fold concentration range of the variable under studv. The K,, values were calculated using the reciprocal plot method of Lineweaver and Burk (1934). RESULTS ASD DISCUSSION
All concentrations shown in the figures and tables are the final concentrations of the compound in the reaction mixture. The results are the average of at least two separate trials. It has been established that P. berghei and P. Zophurae have LDH’s that are electrophoretically distinct from that found in the host erythrocyte (Sherman 1961, 1962). Using polyacrylamide gel (disc) electrophoresis we have confirmed the fact that P. berghei LDH is a distinct electrophoretic entity (Fig. 1). However, in contrast to the work of Sherman (1961, 1962) it was observed that the parasite enzyme could not be detected in the erythrocyte of the infected animal. The LDH of the normal mouse erythrocyte was found to separate into four distinct isozymes but no attempt was made to compare the relative activities of the isozymes on a quantitative basis. The LDH of P. berghei was found to be stereospecific for the L( + ) isomer of lactic acid. The D ( - ) isomer showed no activity when corrections were made for the 3.3% L ( + ) isomer contamination. Similar results were observed with mouse ervthrocvte LDH. While the apparent K,, values for lactic acid, NAD, or NADH showed no major differences, the parasite LDH had a K, value for pyruvate that was 1/36th that of the host cell enzyme (Table I). This indicates that the parasite enzyme has a greater affinity for pyruvate than does the host cell enzvme. P. berghi LDH also eshibited
40
PHISPHtJhiVIDHI
AND
LANGER
ORIGIN
/
J 0 tracings resulting from the scan of polyacrylamide electrophoretic gels stained FIG. 1. Densitometer specifically for LDH. (A = extract of normal mouse erythrocytes; B = extract of infected mouse erythrocytes; C = extract of host cell-free I’. herghei. )
TABLE I Michaelis Constants (K,) for the LDH in CellFree Extracts of Host Cell-Free P. berghei and Normal Mouse Erythrocyte@ K, Substrate or cofactor tested L( +)
acid” Pyruvic NADd NADH”
TABLE II Activity of the LDH in Cell-Free Extracts of host Cell-Free P. berghei and Normal Mouse Eythrocytesa Specific activity of the LDH of
values for the LDH of
P. berghei
Normal mouse erythrocvtes Substrate
Lactic acidr
Specific
1.1 5.8 3.1 2.0
x x x x
lo-2M lo-sM lo-4M lo-4M
1.4 1.6 5.9 1.3
x x x x
lOWeM lo-“M lo-“M lo-4M
a The values were determined as described in Materials and Methods. b L( +) lactic acid concentration range tested, 3.4 x 10-a-3.4 x lo--‘M; NAD concentration, 3.7 x lo-“M. c Pyruvic acid concentration range tested, 1.7 x 10-a-1.7 x lo-GM; NADH concentration, 6.9 x lo-4M. * NAD concentration range tested, 3.7 X lo-s3.7 x lo-5M; L( +) lactic acid concentration, 1.7 x lOWaM. (’ NADH concentration range tested, 6.9 x 10-4-1.7 x IO-sM; pyruvic acid concentration, 1.7 x lo-“M.
higher specific activities for the substrates tested than did the host cell LDH; specifically, 6 times greater for lactic acid and 15 times greater for pyruvic acid ( Table II ) . It is interesting to note that the apparent K, value for pyruvate of the LDH of P.
L( + ) Lactic Pyruvic acid
acid
P. berghei
Normal mouse erythrocytes
0.38” 2.8~
0.06” 0.18’
a The values were determined as described in Materials and Methods. b Fmoles NAD reduced/mg. active protein/minute; L( +) lactic acid concentration, 1.7 x lo-2M; NAD concentration, 3.7 X lo-:iM. L’ kmoles NADH oxidized/mg active protein/ min; pyruvic acid concentration, 1.7 x lo-aM; NADH concentration, 6.9 x lo--‘M.
Zophurue (1.9 x lo-” M) Sherman 1961) is of the same order of magnitude as was the value determined for P. berghei (5.8 x lo-” M) . Duck erythrocytic LDH has a K, value for pyruvate of 1.7 x 106” M, not significantly different from that observed for the P. Zophurae LDH (Sherman 1961) thereby showing species differences in the host-parasite relationship when compared to the data presented in this communication for P. berghei and the mouse erythrocyte. Sherman (1961) also noted that P. lophurae LDH was 2.7 times as active as the duck
MALARIAL
PARASITE
erythrocytic LDH with pyruvic acid as substrate, further pointing out species differences in the host-parasite relationship. In view of the fact that the affinity for pyruvate and the specific activity with pyruvate as substrate was greater for the parasite enzyme than it was for the host cell enzyme, the conclusion that the parasite could effectively remove pyruvate from the host cell for its own use, seems logical. Since pyruvate and lactate are the end products of glycdytic carbohydrate metabolism and it is generally conceded that the erythrocyte does not contain an active citric acid cycle, it appears that the parasite would not interfere with erythrocyte metabolism by removing erythrocytic pyruvate. However, because of the accumulation of lactate resulting from the parasite metabolism of its own and “stolen” pyruvate, a decrease in erythrocytic intracellular pH would be expected, assuming the lactate to be ready diffusible across the parasite cell membrane. The specific effects of this drop in pH on the life span and metabolism of the host erythrocyte and on the in viva development of the parasite is unknown. However, Trager (1953, 1957) noted that the cultivation of P. lophurae was most successful at pH 6.8-6.9. Whether the same can be said about the development of P. berghei has not been investigated. It seems logical to postulate that the higher affinity and activity of parasite LDH for pyruvate results in the accumulation of excess lactate that may be necessary for the maintenance of a slightly acid pH required for the development of the malaria parasite.
41
METABOLISM
ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mrs. Duangduen Jiampermpoon and Mr. Mood Saengsri. REFEHENCES BERGMEYER, H. U., BERNT, E., AND HESS, B. 1963. Lactic dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed. ), pp. 736-741. Academic Press, New York. HOHORST, H. J. 1963. L( +) lactate. Determination with lactic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer. ed. ), pp. 266-270. Academic Press, New York. LANCER, B. W., JR., PHISPHUMVIDHI, P., AXD FRIEDLANDER, Y. 1967. Malarial parasite metabolism: The pentose cycle in Plasmodium berghei. Experimental Parasitology 20, 68-76. LINEWEAVER, H. AND BURK, D. 1934. The determination of enzyme dissociation constants. Journal of the American Chemical Society 56, 658-666. SHERMAN, I. W. 1961. Molecular heterogeneity of lactic dehydrogenase in avian malaria (Plasmodium lophurae) . Journal of Experimental Medicine 114, 1049-1062. SHERMAN, I. W. 1962. Heterogeneity of lactic dehydrogenase in intraerythrocytic parasites. Transactions of the New York Academy of Sciences Ser. II, 24, 944-953. 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. TRACER, W. 1953. Further studies on the extracellular cultivation of an avian malaria parasite. Annals of the Nezc; York Academy of Sciences 56, 1074-1093. TRAGER, W. 1957. The nutrition of an intracellular parasite (avian malaria). Acta Tropica 14, 289-301.