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Isocitric dehydrogenase in unembryonated eggs of Ascaris lumbricoides
EXPERIMEXTAL
PARASITOLOGY
Isocitric
14,
(1963)
Dehydrogenase Ascaris Hiroshi
Department
186-192
of Physiology,
Oya,”
School
in Unemhryonated lumbricoidesl
L. C. Costello,
of Pharmacy,
(Submitted
for
and
University
publication,
W.
Eggs
Smith”
of Maryland, 14 December
of
Baltimore,
Maryland
1962)
Using dialyzed homogenates of unembryonated eggs of Ascaris Zumbricoides var. suum, isocitric dehydrogenase activity was demonstrated. Isocitrate as substrate stimulated oxygen consumption and formation of a-ketoglutarate, In addition, isocitrate stimulated the reduction of 2,6-dichlorophenolindophenol. In all cases, added triphosphopyridine nucleotide (TPN) was required as cofactor for the dehydrogenase activity. Diphosphopyridine nucleotide (DPN) had no such stimulatory effect. The enzymatic activity was further stimulated by the presence of phenazine methosulfate which oxidized TPNH. The addition of pyruvate appeared to stimulate the formation of a-ketoglutarate from isocitrate and oxidized TPNH resulting from the dehydrogenase activity. The results suggested the possibility of a coupling reaction for the oxidation of TPNH. The possible role of isocitric dehydrogenase and TPN in the metabolism of developing eggs was discussed as well as the comparative relationship to the enzyme observed in adult muscle preparations.
from adult muscle (Oya et al., 1962; Oya Costello and Brown (1962) presented pre- and Kikuchi, unpublished data). This presliminary evidence of the presence of iso- ent report is concerned with the further citric dehydrogenase activity in homoge- identification of isocitric dehydrogenase in Ascaris eggs and the cofactor requirements nates of unembryonated Ascaris eggs. Of further interest was the report of Seidman of the enzyme. and Entner (1961) in which the authors were MATERIALS AND METHODS unable to demonstrate the presence of this Unembryonated eggs of Ascaris lumbrienzyme in an adult muscle sarcosomeprepcoides var. suum were harvested and homogaration. Contrary to this, Oya and his enates prepared as previously described colleagues have reported the presence of (‘Costello, 1961, 1963). In addition to other isocitric dehydrogenase activity requiring advantages of this method cited by the TPN4 as a cofactor in particulate extracts author, no unfertilized eggs were present in 1 This investigation was supported by National the preparation as the harvesting technique Science Foundation Research Grant G-23313. ruptured such cells. Essentially all eggs were 2 On leave from Department of Pharmacology, in the one-cell stage. Generally, the homogeJuntendo University School of Medicine, Hongo, nates were prepared in 0.05 A4 potassium Tokyo, Japan. Present position: Research Assophosphate buffer (pH 7.4)) diluted to apciate, Department of Physiology. a Pre-doctoral trainee, P.H.S. Grant DT-46, Deproximately 60% concentration (volume partment of Histology and Embryology, School Dentistry. 4 Abbreviations used: DPN (diphosphopyridine nucleotide) , TPN (triphosphopyridine nucleotide)
of TPNH
(reduced TPN), a-KG (alpha Ph. MS (phenazine methosulfate), (2,6-dichlorophenolindophenol) .
rate), ,
dye
ketoglutaand 2,6-
ISOCITRIC
DEHYDROGENASE
packed eggs/volume buffer), and dialyzed overnight against the same buffer. Dialysis was conducted with two changes of buffer and continuous stirring, with the entire procedure performed in a cold room at approximately 4°C. Manometric experiments were conducted by conventional Warburg techniques using 17 ml (approx.) single sidearm flasks at 37’C. The complete system included 10 ElmolesMgCIB, 3 pmoles MnC12, 50 pmoles nicotinamide, 2.0 ml egg homogenate, 20 pmoles isocitrate, 0.2 pmole TPN or DPN, 0.15 mg phenazine methosulfate, and 0.1 M phosphate buffer (pH 7.4) to adjust the final volume in the main well to 3.0 ml; and 0.2 ml 20% KOH in the alkali well. All flasks were set up in triplicate and were equilibrated for 10 minutes, at which time the contents of the sidearm (isocitrate, TPS or DPN, and phenazine methosulfate) were tipped into the main well. Depending upon the experiment involved, the incubation period was 60 or 90 minutes, at which time the reaction was terminated by the addition of 0.3 ml 60% perchloric acid. The contents were removed and centrifuged at approximately 12,000g for 30 minutes. The supernatant fluid was neutralized with KOH in ice, centrifuged to remove the perchlorate, and analyzed for products. Because of the limiting amount of enzyme activity of the homogenate preparations and the sensitivity of the assays, the contents from identical flasks (triplicates) were combined to determine ketoacid formation. The production of alpha ketoacids was identified as the 2,4-dinitrophenylhydrazones according to the method of Kun and GarciaHernandez (1957). The hydrazones were separated chromatographically with n-butanol-ethanol-water (5 : 1:4). In one experiment (Table VI) incubation of the reaction mixture was performed in glass-stoppered Erlenmeyer flasks ( 125 ml) with the use of a Dubnoff metabolic shaker,
IN
Ascaris
187
instead of Warburg flasks. The complete system included 40 pmoles isocitrate, 0.4 pmole TPN, 20 pmoles MgC12, 6 ymoles MnC12, 100 pmoles nicotinamide, 30 p.moles pyruvate, and 5.0 ml homogenate. The incubation proceeded for 60 minutes at which time perchloric acid was added followed by treatment as described above. During the latter phase of this investigation, modifications of the homogenate preparations were employed which successfully permitted the useof spectrophotometric methods. The homogenates (approx. 30% cont. v/v) were centrifuged at 400g for 5 minutes. The supernatant fluid was removed and dialyzed for 4 hours against phosphate buffer (0.05 M, pH 7.4) with hourly changes and continuous stirring. The dialyzed homogenate was then assayed spectrophotometrically for isocitric dehydrogenase activity. The enzyme activity was determined by the reduction of 2,6-dichlorophenolindopheno1 assayed at 600 ml” with a recording spectrophotometer (Perkin-Elmer Model 350). The complete system contained 50 pmoles potassium phosphate buffer (pH 7.4), 10 ymoles MgC12, 20 pmoles isocitrate, 0.2 pmole 2,6 dye, 0.1 pmole DPN or TPN, 0.2 ml homogenate, and distilled water to adjust the volume to 3.0 ml. The specific activities (mpmoles per hour per milligram protein) were derived from initial lo-minute changes during which time the activity was essentially linear. Protein determinations were according to the method of Lowry et al. ( 1951) The preparations utilized in these spectrophotometric assays generally contained 12-l 7 mg protein per milliliter homogenate. The oxidation and reduction of TPN was assayed directly by observing changes in 340 rnp absorption. The system employed was essentially the same as described above except for the addition of 0.2 pmole of TPN and the elimination of 2,6-dye.
188
OYA,
COSTELLO,
RESULTS
The results of the effects of isocitrate and TPN on oxygen consumption and a-KG formation in egg homogenate are presented in Table I. In the presence of TPN, isocitrate stimulated both oxygen uptake and a-KG formation. The absence of TPN resulted in a marked reduction of this stimulation. In addition, the presence of TPN with Ph. MS stimulated oxygen consumption but not the production of a-KG. In Table II are presented the results concerned with the effects of DPN on the stim-
Efect
of Isocitrate
SMITH
ulation of oxygen consumption and u-KG formation in egg homogenate with isocitrate as substrate. In all cases no a-KG formation was observed and DPN only slightly stimulated the oxygen uptake. A comparison of the effects of TPX and DPN on the stimulation of oxygen consumption and a-KG formation is presented in Table III. TPN markedly stimulated both oxygen uptake and u-KG production by egg homogenates with isocitrate as substrate. In contrast, DPN resulted in no stimulation above the endogenous level.
TABLE I and TPN on Oxygen Consumption Formation in Egg Homogenate
and u-Ketoglutaratr u-Ketogiutarate formed (pmoles)
Oxygen consumed (umoles)
System Isocitrate TPN + Isocitrate
AND
+ TPN + Ph. MS + Ph. MS
Ph. MS
3.82 (1.27 2 0.17)n 2.02 (0.68 k 0.18) 0.43 (0.14 2 0.09)
1.32 nil nil
a The values in parentheses are the mean and S.D. of the flasks in triplicate. All other as the sum of three flasks comprising each system. The incubation period was 90 minutes.
Effect
of Isocitrate
TABLE II and DPN on Oxygen Consumption Formation in Egg Homogenate
+ DPN + Ph. MS + Ph. MS
Ph. MS
Comparison
of the Eflects
System Isocitrate Isocitrate Isocitrate
cl-Ketogiutarate formed (pmoles)
+ TPN + Ph. MS + DPN + Ph. MS + Ph. MS
nil nil nil
3.25 (1.08 k 0.05)cL 2.62 (0.87 k 0.09) 2.52 (0.84 2 0.08)
a The values in parentheses are the mean and as the sum of three flasks comprising each system.
of
are reported
and cl-Ketoglutarate
Oxygen consumed ( ymoles)
System Isocitrate DPN + Isocitrate
values
S.D. of the flasks in triplicate. .-\I1 other The incubation period was 90 minutes.
T.4BLE III TPN and DPN on Oxygen Consumption Formation in Egg Homogenate Oxygen consumed (umoles)
values
are reported
atzd cl-Krtoglutauate a-Ketoglutarate formed (umoles)
11.19 (3.73 2 0.07)” 1.96 (0.65 -t 0.11) 1.85 (0.62 31 0.13)
a The values in parentheses are the mean and S.D. of the flasks in triplicate. rlil other values as the sum of three flasks comprising each system. The incubation period was 90 minutes.
4.61 0.04 0.05 are reported
ISOCITRIC
Effects
of TPN
DEHYDROGENASE
+ +
TPN TPN
+
21.16 3.47 2.68 14.71 2.79
Ph. MS
(7.02 (1.16 (0.90 (4.90 (0.93
f ir & f k
a The values in parentheses are the mean and S.D. of the flasks as the sum of three flasks comprising each system. The incubation
The effects of TPN and Ph. MS on oxygen and a-KG formation in egg homogenates are presented in Table IV. The presence of isocitrate, TPN, and Ph. MS afforded the greatest stimulation of both oxygen uptake and a-KG production. Furthermore, Ph. MS markedly stimulated the oxygen consumption of homogenate in the presence of TPN but resulted in no a-KG formation. TPN in the presence of isocitrate only slightly stimulated oxygen uptake but resulted in the formation of a-KG as well. In the absence of isocitrate no stimulation of a-KG formation was detected. Figure 1 illustrates the marked stimulation of oxygen consumption of homogenates in the presence of isocitrate, TPN, and Ph. MS. In the absence of isocitrate, a decrease in oxygen uptake was observed. Furthermore, the stimulation of respiration by the complete system was immediate and occurred throughout the duration of the experiment. The results presented in Table V demonstrate the spectrophotometric identification consumption
of DPN
and TPN
on Isocitric
System + +
Expt. DPN TPN
and a-Ketoglutarate a-Ketoglutarate formed ( ymoles)
1
a The specific activities were determined are corrected for endogenous activities.
in triplicate. All other period was 90 minutes.
values
are reported
TIME (Minutes) FIG. 1. The relationship of phenazine methosulfate on the of dialyzed egg homogenate. 1, Ph. MS; 2, TPN + Ph. MS; 4, TPN; 5, endogenous control.
Activity
activity
26 20 46 from
8.33 0.85 0.13 0.10 0.10
1%
Expt.
12 12 22
O.lO)a 0.26) 0.05) 0.10) 0.10)
of isocitric dehydrogenase based on the reduction of 2,6-dye. TPN stimulated activity,
TABLE V Dehydrogenase Specific
Isocitrate Isocitrate Isocitrate
189
Oxygen consumed ( pmoles)
Ph. MS
Effects
Ascaris
TABLE IV Methosulfate on Oxygen Consumption Formation in Egg Homogenate
and Phenazine
System Isocitrate Isocitrate Isocitrate TPS + TPN
IN
the reduction
of
Egg Homogenates
(mpmoles/hour/mg 2
isocitrate, TPN, and oxygen consumption Isocitrate + TPN + 3, isocitrate + TPN;
protein)a Expt.
3
Avg.
12 17 38 of 2&dichlorophenolindophenol.
16.6 16.3 35.3 All
values
190
OYA,
COSTELLO,
whereas DPN appeared to have no effect. The average specific activity in the presence of TPN when corrected for endogenous activity was 18.7 mpmoles per hour per milligram protein. The specific activities ascertained for such crude preparations should be considered as approximate with accurate reproducibility difficult. In Table VI are presented the effects of added pyruvate on o-KG formation by egg TABLE VI Efect of Pyruvate on Stimulation of Zsocilk Dehydrogenase Activity in Egg Homogenate a-Ketoglutarate formed (umoles/hour)
System Isocitrate + TPN + Pyruvate Isocitrate + TPiX TPN + Pyruvate
2 so 1.40 0.10
homogenate in the presence of isocitrate and TPN. Pyruvate markedly stimulated the formation of a-KG in the complete system. In the absence of pyruvate, the stimulation of a-KG formation by isocitrate and TPN was reduced. Coinciding with this stimulation was a decrease in the amount of pyruvate
0.5-
-no pyruvote added .....llllll,,,l,lpyruvate added inltiolly -----pyruvote
added
with
at designated
isocitrote point
0.4s f-20.30 g 0.2O.l-
0
I
2
I
Time
I
8
4
I
IO
(minttes)
FIG. 2. The effects of isocitrate and pyruvate on the reduction and oxidation of TPN by dialyzed egg homogenate. All systems initially contained homogenate, buffer, TPN, and MgCI,. After the initial period (indicated by solid arrow), either isocitrate or isocitrate and pyruvate was added to the experimental cuvette and water or pyruvate to the controls. In one system pyruvate was added after partial reduction of TPN by isocitrate (indicated by broken arrow).
AND
SMITH
(2.5 pmole difference) remaining in the system at termination of the incubation period. Figure 2 demonstrates the effects of isocitrate and pyruvate on the reduction and oxidation of TPN. The addition of isocitrate markedly reduced TPN, thereby further demonstrating the presence of dehydrogenase activity. The addition of pyruvate to the system resulted in immediate oxidation of the TPNH. DISCUSSION
In earlier work concerned with the preparation of homogenates of dscaris eggs and the preliminary identification of isocitric dehydrogenase activity, only fresh homogenates were employed (Costello and Brown, 1962). As discussed at that time, concentrated preparations were necessary and would be expected to contain a high amount of endogenous materials which apparently often prevented stimulation of activity by added substances. During the early phases of the present investigation, additional experiments were attempted with the use of undialyzed homogenates. Only slight and often inconsistent stimulation of respiration in experimental systems was observed. With these circumstances in mind, dialyzed homogenates were employed and provided more useful preparations than the undialyzed materials. Such a modification obviously could be applied only to the investigation of enzymes stable enough to withstand the dialysis procedure. In addition, dialysis apparently resulted in the removal or inactivation of cofactors so that such enzyme requirements could be investigated. The results of this investigation demonstrated the presence of isocitric dehydrogenase in dialyzed homogenates of unembryonated Ascaris eggs. The evidence supporting this conclusion was based on the stimulation of oxygen consumption, the formation of a-KG, the reduction of 2,6-dye, and the reduction of TPN by homogenates in the presence of
ISOCITRIC
DEHYDROGENASE
isocitrate and necessary cofactors. These observations corroborated the earlier report of the presence of isocitric dehydrogenase in fresh homogenates (Costello and Brown, 1962). The specific activity of the dialyzed homogenates, based on the reduction of 2,6dye, was calculated to be approximately 35 mumoles per hour per milligram protein. In dialyzed preparations, isocitric dehydrogenase activity was demonstrable only in the presence of added TPN. Furthermore, DPN could not be substituted for TPN. The lack of stimulation by added DPN suggested that this coenzyme was not required for isocitric dehydrogenase activity. Stimulation by TPN was taken as evidence for its requirement for the dehydrogenase activity. This stimulation might possibly have resulted from a recycling of endogenous DPN as follows :
However, the lack of stimulation by added DPN would appear to eliminate this possibility. The addition of Ph. MS to the system resulted in a stimulation of isocitric dehydrogenase activity (Table IV). Apparently Ph. MS was utilized for the oxidatio’n of TPNH. It is important to note, however, that Ph. MS stimulated the oxygen uptake of homogenate in the presence of TPN alone, which did not result in the formation of a-KG. The addition of isocitrate further increased oxygen consumption and stimulated a-KG production as well. Furthermore, a relatively low endogenous oxidation of TPN reduced by isocitrate activity existed and resulted in a slight formation of a-KG. This slight endogenous activity suggested that the homogenate contained a system for oxidizing TPNH which could possibly be related to an electron transport system or a coupling reaction.
IN
AsCUf’is
191
In this regard, it was interesting to observe the marked stimulation of isocitric dehydrogenase activity upon the addition of pyruvate. The concurrent increased formation of a-KG and apparent utilization of pyruvate suggested the possibility of a coupling reaction. Further evidence of this was provided by the immediate oxidation of TPNH upon addition of pyruvate to the system initially containing isocitrate and TPN. Further studies concerning the possible coupling of TPN with isocitrate dehydrogenase and pyruvate dismutation are necessary before this relationship can be conclusively established. The relatio’nship of isocitric dehydrogenase and TPN could conceivably be an important reaction involved in the regulation and direction of some pathways of metabolism in Ascaris eggs. The oxidation of TPNH could result from electron transport activity and/or coupling reactions. It would be of significance to ascertain which relationships exist in Ascaris eggs. However, the elucidation of such information, particularly in Ascaris eggs, poses difficult problems. Perhaps the use of specific inhibitors of the electron transport system will provide such information. This question is raised in view of the obvious changes in metabolism during Ascaris development, particularly in regard to carbohydrate and lipid metabolism (Passey and Fairbairn, 1955, 1957). From a comparative point of view the findings reported here bear an interesting relationship to those observed in adult Ascaris preparations. The identification of isocitric dehydrogenase has now been reported in both the egg stage and adult muscle. Of added interest is the similarity of the coenzyme requirements which, in the egg and adult preparation (Oya et aZ., 1962 ; Oya and Kikuchi, unpublished data), involves TPN. A comparison of &citric dehydrogenase activity during different stages of development of eggs is currently under investigation.
192
OYA,
COSTELLO,
ACKNOWLEDGMENT
The authors wish to express their sincere appreciation for the cooperation and assistance provided by the personnel of the Baltimore Branch of the Schluderberg-Kurdle Company in supplying the ascarids used for this investigation. REFERENCES
COSTELLO, L. C. 1961. A simplified method of isolating Ascaris eggs. Journal of Parasitology 47, 24. COSTELLO, L. C. 1963. FAD extracted from unembryonated Ascan’s lumbricoides eggs. rlccepted for publication in Expevimental Parasitology. COSTELLO, L. C., AND BROWN, H. 1962. Aerobic metabolism of unembryonated eggs of Ascaris lumbricoides. Experimental Parasitology 12, 3340. KUN, E., AND GARCIA-HERSANDEZ, M. 1957. Identification and quantitative determination of keto acids by paper chromatography. Biockemica et Biopkysica Acta 23, 181-186.
AND
SMITH
0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurements with the Folin phenol reagent. Journal of Biochemistry 193, 265-275. OYA, H., AND KIKUCHI, G. Unpublished data. LOWRY,
OYA, H., KIKUCHI, G., HAYASHI, H., AND BANDO, T. 1962. The occurrence of the tricarboxylic acid cycle in the muscle of Ascaris lumbricoides. Japanese Journal of Parasitology. In press. PASSEY, R. F., AND FAIRBAIRN, D. 1955. The respiration of Ascaris lumbvicoides eggs. Canadian Journal of Biochemistry and Physiology, 33, 1033-1046. PASSEY, R. F., AND FAIRBAIRN, D. 1957. The conversion of fat to carbohydrate during embryonation of ascaris eggs. Canadian Journal of Biochemistry and Physiology 36, 511-525. SEIDMAN, I., AND ENTNER, N. 1961. Oxidative enzymes and their role in phosphorylation in sarcosomes of adult Ascaris lumbricoides. The Journal of Biochemistry 236, 915-919.
PARASITOLOGY
Isocitric
14,
(1963)
Dehydrogenase Ascaris Hiroshi
Department
186-192
of Physiology,
Oya,”
School
in Unemhryonated lumbricoidesl
L. C. Costello,
of Pharmacy,
(Submitted
for
and
University
publication,
W.
Eggs
Smith”
of Maryland, 14 December
of
Baltimore,
Maryland
1962)
Using dialyzed homogenates of unembryonated eggs of Ascaris Zumbricoides var. suum, isocitric dehydrogenase activity was demonstrated. Isocitrate as substrate stimulated oxygen consumption and formation of a-ketoglutarate, In addition, isocitrate stimulated the reduction of 2,6-dichlorophenolindophenol. In all cases, added triphosphopyridine nucleotide (TPN) was required as cofactor for the dehydrogenase activity. Diphosphopyridine nucleotide (DPN) had no such stimulatory effect. The enzymatic activity was further stimulated by the presence of phenazine methosulfate which oxidized TPNH. The addition of pyruvate appeared to stimulate the formation of a-ketoglutarate from isocitrate and oxidized TPNH resulting from the dehydrogenase activity. The results suggested the possibility of a coupling reaction for the oxidation of TPNH. The possible role of isocitric dehydrogenase and TPN in the metabolism of developing eggs was discussed as well as the comparative relationship to the enzyme observed in adult muscle preparations.
from adult muscle (Oya et al., 1962; Oya Costello and Brown (1962) presented pre- and Kikuchi, unpublished data). This presliminary evidence of the presence of iso- ent report is concerned with the further citric dehydrogenase activity in homoge- identification of isocitric dehydrogenase in Ascaris eggs and the cofactor requirements nates of unembryonated Ascaris eggs. Of further interest was the report of Seidman of the enzyme. and Entner (1961) in which the authors were MATERIALS AND METHODS unable to demonstrate the presence of this Unembryonated eggs of Ascaris lumbrienzyme in an adult muscle sarcosomeprepcoides var. suum were harvested and homogaration. Contrary to this, Oya and his enates prepared as previously described colleagues have reported the presence of (‘Costello, 1961, 1963). In addition to other isocitric dehydrogenase activity requiring advantages of this method cited by the TPN4 as a cofactor in particulate extracts author, no unfertilized eggs were present in 1 This investigation was supported by National the preparation as the harvesting technique Science Foundation Research Grant G-23313. ruptured such cells. Essentially all eggs were 2 On leave from Department of Pharmacology, in the one-cell stage. Generally, the homogeJuntendo University School of Medicine, Hongo, nates were prepared in 0.05 A4 potassium Tokyo, Japan. Present position: Research Assophosphate buffer (pH 7.4)) diluted to apciate, Department of Physiology. a Pre-doctoral trainee, P.H.S. Grant DT-46, Deproximately 60% concentration (volume partment of Histology and Embryology, School Dentistry. 4 Abbreviations used: DPN (diphosphopyridine nucleotide) , TPN (triphosphopyridine nucleotide)
of TPNH
(reduced TPN), a-KG (alpha Ph. MS (phenazine methosulfate), (2,6-dichlorophenolindophenol) .
rate), ,
dye
ketoglutaand 2,6-
ISOCITRIC
DEHYDROGENASE
packed eggs/volume buffer), and dialyzed overnight against the same buffer. Dialysis was conducted with two changes of buffer and continuous stirring, with the entire procedure performed in a cold room at approximately 4°C. Manometric experiments were conducted by conventional Warburg techniques using 17 ml (approx.) single sidearm flasks at 37’C. The complete system included 10 ElmolesMgCIB, 3 pmoles MnC12, 50 pmoles nicotinamide, 2.0 ml egg homogenate, 20 pmoles isocitrate, 0.2 pmole TPN or DPN, 0.15 mg phenazine methosulfate, and 0.1 M phosphate buffer (pH 7.4) to adjust the final volume in the main well to 3.0 ml; and 0.2 ml 20% KOH in the alkali well. All flasks were set up in triplicate and were equilibrated for 10 minutes, at which time the contents of the sidearm (isocitrate, TPS or DPN, and phenazine methosulfate) were tipped into the main well. Depending upon the experiment involved, the incubation period was 60 or 90 minutes, at which time the reaction was terminated by the addition of 0.3 ml 60% perchloric acid. The contents were removed and centrifuged at approximately 12,000g for 30 minutes. The supernatant fluid was neutralized with KOH in ice, centrifuged to remove the perchlorate, and analyzed for products. Because of the limiting amount of enzyme activity of the homogenate preparations and the sensitivity of the assays, the contents from identical flasks (triplicates) were combined to determine ketoacid formation. The production of alpha ketoacids was identified as the 2,4-dinitrophenylhydrazones according to the method of Kun and GarciaHernandez (1957). The hydrazones were separated chromatographically with n-butanol-ethanol-water (5 : 1:4). In one experiment (Table VI) incubation of the reaction mixture was performed in glass-stoppered Erlenmeyer flasks ( 125 ml) with the use of a Dubnoff metabolic shaker,
IN
Ascaris
187
instead of Warburg flasks. The complete system included 40 pmoles isocitrate, 0.4 pmole TPN, 20 pmoles MgC12, 6 ymoles MnC12, 100 pmoles nicotinamide, 30 p.moles pyruvate, and 5.0 ml homogenate. The incubation proceeded for 60 minutes at which time perchloric acid was added followed by treatment as described above. During the latter phase of this investigation, modifications of the homogenate preparations were employed which successfully permitted the useof spectrophotometric methods. The homogenates (approx. 30% cont. v/v) were centrifuged at 400g for 5 minutes. The supernatant fluid was removed and dialyzed for 4 hours against phosphate buffer (0.05 M, pH 7.4) with hourly changes and continuous stirring. The dialyzed homogenate was then assayed spectrophotometrically for isocitric dehydrogenase activity. The enzyme activity was determined by the reduction of 2,6-dichlorophenolindopheno1 assayed at 600 ml” with a recording spectrophotometer (Perkin-Elmer Model 350). The complete system contained 50 pmoles potassium phosphate buffer (pH 7.4), 10 ymoles MgC12, 20 pmoles isocitrate, 0.2 pmole 2,6 dye, 0.1 pmole DPN or TPN, 0.2 ml homogenate, and distilled water to adjust the volume to 3.0 ml. The specific activities (mpmoles per hour per milligram protein) were derived from initial lo-minute changes during which time the activity was essentially linear. Protein determinations were according to the method of Lowry et al. ( 1951) The preparations utilized in these spectrophotometric assays generally contained 12-l 7 mg protein per milliliter homogenate. The oxidation and reduction of TPN was assayed directly by observing changes in 340 rnp absorption. The system employed was essentially the same as described above except for the addition of 0.2 pmole of TPN and the elimination of 2,6-dye.
188
OYA,
COSTELLO,
RESULTS
The results of the effects of isocitrate and TPN on oxygen consumption and a-KG formation in egg homogenate are presented in Table I. In the presence of TPN, isocitrate stimulated both oxygen uptake and a-KG formation. The absence of TPN resulted in a marked reduction of this stimulation. In addition, the presence of TPN with Ph. MS stimulated oxygen consumption but not the production of a-KG. In Table II are presented the results concerned with the effects of DPN on the stim-
Efect
of Isocitrate
SMITH
ulation of oxygen consumption and u-KG formation in egg homogenate with isocitrate as substrate. In all cases no a-KG formation was observed and DPN only slightly stimulated the oxygen uptake. A comparison of the effects of TPX and DPN on the stimulation of oxygen consumption and a-KG formation is presented in Table III. TPN markedly stimulated both oxygen uptake and u-KG production by egg homogenates with isocitrate as substrate. In contrast, DPN resulted in no stimulation above the endogenous level.
TABLE I and TPN on Oxygen Consumption Formation in Egg Homogenate
and u-Ketoglutaratr u-Ketogiutarate formed (pmoles)
Oxygen consumed (umoles)
System Isocitrate TPN + Isocitrate
AND
+ TPN + Ph. MS + Ph. MS
Ph. MS
3.82 (1.27 2 0.17)n 2.02 (0.68 k 0.18) 0.43 (0.14 2 0.09)
1.32 nil nil
a The values in parentheses are the mean and S.D. of the flasks in triplicate. All other as the sum of three flasks comprising each system. The incubation period was 90 minutes.
Effect
of Isocitrate
TABLE II and DPN on Oxygen Consumption Formation in Egg Homogenate
+ DPN + Ph. MS + Ph. MS
Ph. MS
Comparison
of the Eflects
System Isocitrate Isocitrate Isocitrate
cl-Ketogiutarate formed (pmoles)
+ TPN + Ph. MS + DPN + Ph. MS + Ph. MS
nil nil nil
3.25 (1.08 k 0.05)cL 2.62 (0.87 k 0.09) 2.52 (0.84 2 0.08)
a The values in parentheses are the mean and as the sum of three flasks comprising each system.
of
are reported
and cl-Ketoglutarate
Oxygen consumed ( ymoles)
System Isocitrate DPN + Isocitrate
values
S.D. of the flasks in triplicate. .-\I1 other The incubation period was 90 minutes.
T.4BLE III TPN and DPN on Oxygen Consumption Formation in Egg Homogenate Oxygen consumed (umoles)
values
are reported
atzd cl-Krtoglutauate a-Ketoglutarate formed (umoles)
11.19 (3.73 2 0.07)” 1.96 (0.65 -t 0.11) 1.85 (0.62 31 0.13)
a The values in parentheses are the mean and S.D. of the flasks in triplicate. rlil other values as the sum of three flasks comprising each system. The incubation period was 90 minutes.
4.61 0.04 0.05 are reported
ISOCITRIC
Effects
of TPN
DEHYDROGENASE
+ +
TPN TPN
+
21.16 3.47 2.68 14.71 2.79
Ph. MS
(7.02 (1.16 (0.90 (4.90 (0.93
f ir & f k
a The values in parentheses are the mean and S.D. of the flasks as the sum of three flasks comprising each system. The incubation
The effects of TPN and Ph. MS on oxygen and a-KG formation in egg homogenates are presented in Table IV. The presence of isocitrate, TPN, and Ph. MS afforded the greatest stimulation of both oxygen uptake and a-KG production. Furthermore, Ph. MS markedly stimulated the oxygen consumption of homogenate in the presence of TPN but resulted in no a-KG formation. TPN in the presence of isocitrate only slightly stimulated oxygen uptake but resulted in the formation of a-KG as well. In the absence of isocitrate no stimulation of a-KG formation was detected. Figure 1 illustrates the marked stimulation of oxygen consumption of homogenates in the presence of isocitrate, TPN, and Ph. MS. In the absence of isocitrate, a decrease in oxygen uptake was observed. Furthermore, the stimulation of respiration by the complete system was immediate and occurred throughout the duration of the experiment. The results presented in Table V demonstrate the spectrophotometric identification consumption
of DPN
and TPN
on Isocitric
System + +
Expt. DPN TPN
and a-Ketoglutarate a-Ketoglutarate formed ( ymoles)
1
a The specific activities were determined are corrected for endogenous activities.
in triplicate. All other period was 90 minutes.
values
are reported
TIME (Minutes) FIG. 1. The relationship of phenazine methosulfate on the of dialyzed egg homogenate. 1, Ph. MS; 2, TPN + Ph. MS; 4, TPN; 5, endogenous control.
Activity
activity
26 20 46 from
8.33 0.85 0.13 0.10 0.10
1%
Expt.
12 12 22
O.lO)a 0.26) 0.05) 0.10) 0.10)
of isocitric dehydrogenase based on the reduction of 2,6-dye. TPN stimulated activity,
TABLE V Dehydrogenase Specific
Isocitrate Isocitrate Isocitrate
189
Oxygen consumed ( pmoles)
Ph. MS
Effects
Ascaris
TABLE IV Methosulfate on Oxygen Consumption Formation in Egg Homogenate
and Phenazine
System Isocitrate Isocitrate Isocitrate TPS + TPN
IN
the reduction
of
Egg Homogenates
(mpmoles/hour/mg 2
isocitrate, TPN, and oxygen consumption Isocitrate + TPN + 3, isocitrate + TPN;
protein)a Expt.
3
Avg.
12 17 38 of 2&dichlorophenolindophenol.
16.6 16.3 35.3 All
values
190
OYA,
COSTELLO,
whereas DPN appeared to have no effect. The average specific activity in the presence of TPN when corrected for endogenous activity was 18.7 mpmoles per hour per milligram protein. The specific activities ascertained for such crude preparations should be considered as approximate with accurate reproducibility difficult. In Table VI are presented the effects of added pyruvate on o-KG formation by egg TABLE VI Efect of Pyruvate on Stimulation of Zsocilk Dehydrogenase Activity in Egg Homogenate a-Ketoglutarate formed (umoles/hour)
System Isocitrate + TPN + Pyruvate Isocitrate + TPiX TPN + Pyruvate
2 so 1.40 0.10
homogenate in the presence of isocitrate and TPN. Pyruvate markedly stimulated the formation of a-KG in the complete system. In the absence of pyruvate, the stimulation of a-KG formation by isocitrate and TPN was reduced. Coinciding with this stimulation was a decrease in the amount of pyruvate
0.5-
-no pyruvote added .....llllll,,,l,lpyruvate added inltiolly -----pyruvote
added
with
at designated
isocitrote point
0.4s f-20.30 g 0.2O.l-
0
I
2
I
Time
I
8
4
I
IO
(minttes)
FIG. 2. The effects of isocitrate and pyruvate on the reduction and oxidation of TPN by dialyzed egg homogenate. All systems initially contained homogenate, buffer, TPN, and MgCI,. After the initial period (indicated by solid arrow), either isocitrate or isocitrate and pyruvate was added to the experimental cuvette and water or pyruvate to the controls. In one system pyruvate was added after partial reduction of TPN by isocitrate (indicated by broken arrow).
AND
SMITH
(2.5 pmole difference) remaining in the system at termination of the incubation period. Figure 2 demonstrates the effects of isocitrate and pyruvate on the reduction and oxidation of TPN. The addition of isocitrate markedly reduced TPN, thereby further demonstrating the presence of dehydrogenase activity. The addition of pyruvate to the system resulted in immediate oxidation of the TPNH. DISCUSSION
In earlier work concerned with the preparation of homogenates of dscaris eggs and the preliminary identification of isocitric dehydrogenase activity, only fresh homogenates were employed (Costello and Brown, 1962). As discussed at that time, concentrated preparations were necessary and would be expected to contain a high amount of endogenous materials which apparently often prevented stimulation of activity by added substances. During the early phases of the present investigation, additional experiments were attempted with the use of undialyzed homogenates. Only slight and often inconsistent stimulation of respiration in experimental systems was observed. With these circumstances in mind, dialyzed homogenates were employed and provided more useful preparations than the undialyzed materials. Such a modification obviously could be applied only to the investigation of enzymes stable enough to withstand the dialysis procedure. In addition, dialysis apparently resulted in the removal or inactivation of cofactors so that such enzyme requirements could be investigated. The results of this investigation demonstrated the presence of isocitric dehydrogenase in dialyzed homogenates of unembryonated Ascaris eggs. The evidence supporting this conclusion was based on the stimulation of oxygen consumption, the formation of a-KG, the reduction of 2,6-dye, and the reduction of TPN by homogenates in the presence of
ISOCITRIC
DEHYDROGENASE
isocitrate and necessary cofactors. These observations corroborated the earlier report of the presence of isocitric dehydrogenase in fresh homogenates (Costello and Brown, 1962). The specific activity of the dialyzed homogenates, based on the reduction of 2,6dye, was calculated to be approximately 35 mumoles per hour per milligram protein. In dialyzed preparations, isocitric dehydrogenase activity was demonstrable only in the presence of added TPN. Furthermore, DPN could not be substituted for TPN. The lack of stimulation by added DPN suggested that this coenzyme was not required for isocitric dehydrogenase activity. Stimulation by TPN was taken as evidence for its requirement for the dehydrogenase activity. This stimulation might possibly have resulted from a recycling of endogenous DPN as follows :
However, the lack of stimulation by added DPN would appear to eliminate this possibility. The addition of Ph. MS to the system resulted in a stimulation of isocitric dehydrogenase activity (Table IV). Apparently Ph. MS was utilized for the oxidatio’n of TPNH. It is important to note, however, that Ph. MS stimulated the oxygen uptake of homogenate in the presence of TPN alone, which did not result in the formation of a-KG. The addition of isocitrate further increased oxygen consumption and stimulated a-KG production as well. Furthermore, a relatively low endogenous oxidation of TPN reduced by isocitrate activity existed and resulted in a slight formation of a-KG. This slight endogenous activity suggested that the homogenate contained a system for oxidizing TPNH which could possibly be related to an electron transport system or a coupling reaction.
IN
AsCUf’is
191
In this regard, it was interesting to observe the marked stimulation of isocitric dehydrogenase activity upon the addition of pyruvate. The concurrent increased formation of a-KG and apparent utilization of pyruvate suggested the possibility of a coupling reaction. Further evidence of this was provided by the immediate oxidation of TPNH upon addition of pyruvate to the system initially containing isocitrate and TPN. Further studies concerning the possible coupling of TPN with isocitrate dehydrogenase and pyruvate dismutation are necessary before this relationship can be conclusively established. The relatio’nship of isocitric dehydrogenase and TPN could conceivably be an important reaction involved in the regulation and direction of some pathways of metabolism in Ascaris eggs. The oxidation of TPNH could result from electron transport activity and/or coupling reactions. It would be of significance to ascertain which relationships exist in Ascaris eggs. However, the elucidation of such information, particularly in Ascaris eggs, poses difficult problems. Perhaps the use of specific inhibitors of the electron transport system will provide such information. This question is raised in view of the obvious changes in metabolism during Ascaris development, particularly in regard to carbohydrate and lipid metabolism (Passey and Fairbairn, 1955, 1957). From a comparative point of view the findings reported here bear an interesting relationship to those observed in adult Ascaris preparations. The identification of isocitric dehydrogenase has now been reported in both the egg stage and adult muscle. Of added interest is the similarity of the coenzyme requirements which, in the egg and adult preparation (Oya et aZ., 1962 ; Oya and Kikuchi, unpublished data), involves TPN. A comparison of &citric dehydrogenase activity during different stages of development of eggs is currently under investigation.
192
OYA,
COSTELLO,
ACKNOWLEDGMENT
The authors wish to express their sincere appreciation for the cooperation and assistance provided by the personnel of the Baltimore Branch of the Schluderberg-Kurdle Company in supplying the ascarids used for this investigation. REFERENCES
COSTELLO, L. C. 1961. A simplified method of isolating Ascaris eggs. Journal of Parasitology 47, 24. COSTELLO, L. C. 1963. FAD extracted from unembryonated Ascan’s lumbricoides eggs. rlccepted for publication in Expevimental Parasitology. COSTELLO, L. C., AND BROWN, H. 1962. Aerobic metabolism of unembryonated eggs of Ascaris lumbricoides. Experimental Parasitology 12, 3340. KUN, E., AND GARCIA-HERSANDEZ, M. 1957. Identification and quantitative determination of keto acids by paper chromatography. Biockemica et Biopkysica Acta 23, 181-186.
AND
SMITH
0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurements with the Folin phenol reagent. Journal of Biochemistry 193, 265-275. OYA, H., AND KIKUCHI, G. Unpublished data. LOWRY,
OYA, H., KIKUCHI, G., HAYASHI, H., AND BANDO, T. 1962. The occurrence of the tricarboxylic acid cycle in the muscle of Ascaris lumbricoides. Japanese Journal of Parasitology. In press. PASSEY, R. F., AND FAIRBAIRN, D. 1955. The respiration of Ascaris lumbvicoides eggs. Canadian Journal of Biochemistry and Physiology, 33, 1033-1046. PASSEY, R. F., AND FAIRBAIRN, D. 1957. The conversion of fat to carbohydrate during embryonation of ascaris eggs. Canadian Journal of Biochemistry and Physiology 36, 511-525. SEIDMAN, I., AND ENTNER, N. 1961. Oxidative enzymes and their role in phosphorylation in sarcosomes of adult Ascaris lumbricoides. The Journal of Biochemistry 236, 915-919.