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Studies on the TCA cycle of Macracanthornchus hirudinaceus (Acanthocephala)
Comp. Biochem. Physiol.,
1966, Vol. 18, pp. 663 to 667. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION STUDIES ON THE TCA CYCLE OF MACRACANTHORHYNCHUS HIRUDINACEUS (ACANTHOCEPHALA)* T. T. D U N A G A N and C. C. S C H E I F I N G E R Department of Physiology, Southern Illinois University, Carbondale, Illinois, U.S.A. (Received 4 February
1966)
Aintract--1. Four tricaxboxylicacid cycle enzymes from female Macracanthorhynchus hirudinaceus (Acanthocephala) from swine were assayed in the mitochondrial pellet and supernatant of this pellet. 2. The specific activity/mg protein indicates that aconitase and isocitric dehydrogenase are not present in detectable quantities. Fumarase is present and malate dehydrogenase activity is comparatively high. 3. A comparison of the data on substrates and on enzymes from Acanthocephala supports the absence of an operational TCA cycle. INTRODUCTION A DECADEago Read (1956) called attention to the fact that we knew very little and understood less about the actual metabolic economy of parasites. He emphasized the need to consider the ecology of the parasite and to get away from the idea that the best system for the parasite is to possess an efficient energy machine. It is now recognized that parasites may possess a number of metabolic "modifications" best suited to their needs and that energy does not always head the list. As expected, our knowledge of carbohydrate metabolism, though far from complete, is still much better understood than all other aspects of parasite physiology. As Bueding & Most (1953) pointed out, the Embden-Meyerhof scheme of glycolysis has been observed in every helminth in which the problem has been studied. As expected, different parasites have varying emphases on the production of lactic acid. Likewise, a number of variations in oxidative phosphorylation as well as the importance of the TCA cycle and related shunts exists. There is little doubt that glycolysis exists in all Acanthocephala. Previously published work on the enzymes of Acanthocephala indicates that MDH, LDH, triose phosphate dehydrogenase and aldolase are present but otherwise not much is known concerning the enzymes involved in the intermediary metabolism of * This study was supported by Public Health Service Research Grant AI-05717 from the National Institutes of Health. 663
664
T. T . DUNAGAN AND C. C. SCI-]~IFINGER
this group. Additional information on substrates has recently been published by Bryant & Nicholas (1965). This study confirms the idea that the TCA cycle, if present (and these results do not indicate that it is), is not a significant contributor to the overall economy of M. hirudinaceus. MATERIALS AND METHODS
Macracanthorhynchus hirudinaceus were obtained from a local packing company and placed in Dewar flasks containing minimal amounts of Tyrodes balanced salt solution previously warmed to 37°C. Upon return to the laboratory, the worms were washed in several changes of 0.07 M KH~PO4 until all adhering material was removed. All work performed following the initial step of washing was carried out in the cold. Occasionally a different buffer was used for the washing depending on the enzyme to be assayed. After washing, the worms were ground into a smooth paste by using 0.5 g of glass beads (0.14 mm in diameter) in a cold mortar. This mixture was washed into a centrifuge tube using the appropriate buffer and centrifuged at: 0°C for 10 min at 490g (7.8 cm Ray); resuspended, 10 min at 350g (7"8 cm Ray); resuspended again, 10 min at 350g (7.8 cm Ray); then, the nuclear pellet discarded and supernatants pooled and spun at 34,800g (7.8 cm Ray) three times, each time discarding the supernatant and resuspending the pellet in fresh sucrose. The pellet from this last run was the mitochondrial fraction used. This isolation procedure is essentially that of Bahr & Zeitter (1962). Isodtric dehydrogenase. The crude enzyme extract previously obtained was further fractionated with ammonium sulfate and ethanol according to the technique of Kornberg & Pricer (1951). The spectrophotometric assay of activity was that of Ochoa (1955) which follows the reduction of NADPH at 340 mtz using DLisocitrate as substrate. One unit of enzyme is that amount of enzyme which causes the reduction of ~M of NADPH in 20 min. Aconitase. The assay procedure of Racker (1950) using 0.33 M sodium citrate or 0.01 M sodium DL-isocitrate to form c~-aconitic acid was used. The spectrophotometric assay at 240 mlz is a measure of the number of unsaturated carbon linkages and has a number of limitations as pointed out by Racker (loc. dr.). One unit of activity has been defined as that amount of enzyme which will cause an initial rate of increase in absorbency at 240 mtz of 0.001/min. Fumarase. This enzyme was also determined by the methods of Racker (1950) except that the final assay mixture contained 0-05 M sodium 1-malate rather than the substrate used for aconitase determinations. Malate dehydrogenase. This enzyme was determined by the technique of Ochoa (1955). This technique was used without modifications and proved satisfactory as written. Protein determination. These were made following the technique of WaddeU (1956). Controls. Throughout the assay of these enzyme systems, control determinations were performed on colonal isolates (F~) of bakers' yeast. These preparations were grown aerobically in 2% glucose complex liquid media for 48 hr for study of
665
TCA CYCLE OF M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
TCA enzymes. It is well known that the levels of activity of the various enzymes can be manipulated according to the desires of the investigator by modifications in growth conditions. The authors have selected this colonal isolate to test the assay system employed and to provide some method of comparison of the activities observed in M . hirudinaceus with a known system. RESULTS AND DISCUSSION Table 1 presents information concerning four enzymes found in the TCA cycle. Of the four examined, M D H was found in high concentration and fumarase was found in small amounts. Aconitase and isocitric dehydrogenase were absent. Current information about this aspect of the metabolism of helminths is sketchy and incomplete. Reviewing the metabolism of cestodes, Read & Simmons (1963) TABLE 1 - - T R I C A R B O X Y L I C
Enzyme
Aconitase Isocitrate dehydrogenase with NADP+ Isocitrate dehydrogenase with NAD+ Fumarase Malate dehydrogenase
ACID CYCLE ENZYMES FROM FEMALE
1}I. hirudinaceus
Control Supematant of Mitochondrial pellet 34,800 g preparation 34,800 g preparation supernatant of 29,510 g preparation (activity/rag (activity/rag (activity/rag protein) protein) protein) 0"6 _+ 0"14
0"6 + 0"04
102"2
0"14+ 0"09
0-26+ 0"04
17"5
0"164- 0"09 2"0 _+ 0"3 1039 _+10
0"27+ 0"08 16"8 _+ 1"1 841 +_ 8
12-7 6"9 4"4
Specific activity based on units defined in Bergmeyer (1963) and mentioned in the Materials and Methods section. Controls were colonal isolates of bakers' yeast (F~.) grown aerobically for 48 hr prior to assay. summarized the evidence for an operative Krebs cycle in cestodes thus: "Evidence for oxidation of intermediates of the tricarboxylic acid cycle is incomplete" and "viewing all evidence available it must be concluded that the presence of a cycle has not been demonstrated". In an earlier review, Read (1961) had listed succinate, fumarate and malate as being metabolized but with negative findings with citrate, isocitrate and ~-ketoglutarate. However, this is not the case with nematodes in which the Krebs cycle has been demonstrated in some and not in others (Fairbairn, 1960). Oya et al. (1965) was unable to demonstrate the TCA cycle in Ascaris lumbricoides using enzyme assay techniques but did succeed in showing that such a system existed by using C 14 labelled intermediates. These authors concluded that although the TCA cycle existed that certain rate limiting steps made it of no practical importance to the parasite. It is also evident that a number of different helminths have what appears to be the last half of the Krebs cycle. As evidenced
666
T . T . D U N A O A N AND C. C. SCHEIFINGER
from Table I, M. hirudinaceusfallsinto the lattercategory. Of the four enzymes presented here, aconitase and isocitric dehydrogcnase received the majority of attention. At no time have we been encouraged to believe that these enzymes are present. Attempts to induce their formation by increasing the oxygen tcnsion (which works well with many single-cellfacultativeanaerobic organisms) of the medium in which they were maintained failed to give positive results. It is also interestingto note that compared to the controls the activityof fumarase is rather low whereas that of M D H is very high. There is no doubt that because of the absence of aconitase and isocitricdehydrogcnase M. hirudinaceusis predominately anaerobic in the type of metabolic systems employed. However, the high M D H activityand low fumarase activityare not readily explained. Bryant & Nicholas (1965), working with Moniliformis dubius, also failed to observe the first half of the Krebs cycle when its intermediates were sought; however, they did suggest that the TCA cycle was operative when there was an excess of one or more of the intermediates of the cycle. This was based on the presence of radioactive glutamate which occurred in small amounts following a long reaction time of 3 hr using a cell-free system. A large glutamate pool is understandable since many amino acids can be degraded to it. For this reason it is possible to find small amounts of radioactive glutamate following a long incubation. We, therefore, do not believe this is sufficient evidence for the presence of these reactions. There is no indication that ~-ketoglutarate and isocitrate were assayed by Bryant & Nicholas (1965). Since these are stable intermediates and are easy to isolate and measure, one would have expected some information on their presence or absence. Additional information may also have been obtained from a stepwise incubation. If the time of incubation was increased in increments of 10-15 min per mixture, an increase in products formed per increase in time should result. Had this been done, one could possibly tell the order in which the products are formed and obtain some idea of the sequence of events which takes place. This would uncover any radioactive compound occurring by the degradation of an intermediate formed earlier such as the formation of glutamate from the degradation of 1-ornithine, histidine, proline or arginine. From our own information we do not know if the enzyme substrates are present in the parasite. However, since we were unable to detect the enzymes by the assay systems employed, if the substrate is present the enzymes must not be inducible. We believe that the data of Bryant & Nicholas (1965) supports the enzyme data presented here. It would therefore appear that Acanthocephala do not possess a functional TCA system in the classical sense. REFERENCES BAHRG. F. & ZEITTERE. (1962) Study of mitochondria in rat liver. ~. Cell Biol. 15, 489-501. BEROMEYER HANS-ULRICH, Editor (1963) Methods of Enzymatic Analysis. Academic Press, New York. BRYANT C. & NICHOLASW. L. (1965) Intermediary metabolism in Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 15, 103-112.
TCA CYCLE OF M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
667
BUEDING E. & MOST H. (1953) Helminths: Metabolism, nutrition, and chemotherapy. A. Rev. Microbiol. 7, 295-326. FAIimAmN D. (1960) The physiology and biochemistry of Nematodes. In Nematology (Edited by SASSEI~J. N. & JENKINS W . R.). Univ. of North Carolina Press. KORNBEROA. & PRICERW. E. JR. (1951) Di- and triphosphopyridine nucleotide isocitric dehydrogenases in yeast, d~. b/ol. Chem. 189, 123-136. OCHOA S. (1955) Malic dehydrogenase from pig heart. In Methods in Enzymology, Vol. 1 (Edited by COLOWICKS. P. • KAPLANN. O.). Academic Press, New York. OYA H, KIK'dCHI G, BANDOT. & HAYASHIH. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides var. su/s. Expl Parasitol. 17, 229-240. RACKERE. (1950) Spectrophotometric measurement of the enzymatic formation of fumarase and ds-aconitic acids. Biochera. biophys. Acta 4, 211-214. READ C. P. (1961) The carbohydrate metabolism of worms. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTINA. W.). Univ. of Washington Press, Seattle, Washington. READ C. P. (1956) Carbohydrate metabolism of Hymenolepis diminuta. Expl Parasitol. 5, 325-344-. READC. P. & SIMMONSJ. E. JR. (1963) Biochemistry and physiology of tapeworms. Physiol. Rev. 43, 263-305. WADDELLW. J. (1956) A simple ultraviolet spectrophotometric method for the determination of protein..7. Lab. clin. Med. 48, 311-314.
1966, Vol. 18, pp. 663 to 667. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION STUDIES ON THE TCA CYCLE OF MACRACANTHORHYNCHUS HIRUDINACEUS (ACANTHOCEPHALA)* T. T. D U N A G A N and C. C. S C H E I F I N G E R Department of Physiology, Southern Illinois University, Carbondale, Illinois, U.S.A. (Received 4 February
1966)
Aintract--1. Four tricaxboxylicacid cycle enzymes from female Macracanthorhynchus hirudinaceus (Acanthocephala) from swine were assayed in the mitochondrial pellet and supernatant of this pellet. 2. The specific activity/mg protein indicates that aconitase and isocitric dehydrogenase are not present in detectable quantities. Fumarase is present and malate dehydrogenase activity is comparatively high. 3. A comparison of the data on substrates and on enzymes from Acanthocephala supports the absence of an operational TCA cycle. INTRODUCTION A DECADEago Read (1956) called attention to the fact that we knew very little and understood less about the actual metabolic economy of parasites. He emphasized the need to consider the ecology of the parasite and to get away from the idea that the best system for the parasite is to possess an efficient energy machine. It is now recognized that parasites may possess a number of metabolic "modifications" best suited to their needs and that energy does not always head the list. As expected, our knowledge of carbohydrate metabolism, though far from complete, is still much better understood than all other aspects of parasite physiology. As Bueding & Most (1953) pointed out, the Embden-Meyerhof scheme of glycolysis has been observed in every helminth in which the problem has been studied. As expected, different parasites have varying emphases on the production of lactic acid. Likewise, a number of variations in oxidative phosphorylation as well as the importance of the TCA cycle and related shunts exists. There is little doubt that glycolysis exists in all Acanthocephala. Previously published work on the enzymes of Acanthocephala indicates that MDH, LDH, triose phosphate dehydrogenase and aldolase are present but otherwise not much is known concerning the enzymes involved in the intermediary metabolism of * This study was supported by Public Health Service Research Grant AI-05717 from the National Institutes of Health. 663
664
T. T . DUNAGAN AND C. C. SCI-]~IFINGER
this group. Additional information on substrates has recently been published by Bryant & Nicholas (1965). This study confirms the idea that the TCA cycle, if present (and these results do not indicate that it is), is not a significant contributor to the overall economy of M. hirudinaceus. MATERIALS AND METHODS
Macracanthorhynchus hirudinaceus were obtained from a local packing company and placed in Dewar flasks containing minimal amounts of Tyrodes balanced salt solution previously warmed to 37°C. Upon return to the laboratory, the worms were washed in several changes of 0.07 M KH~PO4 until all adhering material was removed. All work performed following the initial step of washing was carried out in the cold. Occasionally a different buffer was used for the washing depending on the enzyme to be assayed. After washing, the worms were ground into a smooth paste by using 0.5 g of glass beads (0.14 mm in diameter) in a cold mortar. This mixture was washed into a centrifuge tube using the appropriate buffer and centrifuged at: 0°C for 10 min at 490g (7.8 cm Ray); resuspended, 10 min at 350g (7"8 cm Ray); resuspended again, 10 min at 350g (7.8 cm Ray); then, the nuclear pellet discarded and supernatants pooled and spun at 34,800g (7.8 cm Ray) three times, each time discarding the supernatant and resuspending the pellet in fresh sucrose. The pellet from this last run was the mitochondrial fraction used. This isolation procedure is essentially that of Bahr & Zeitter (1962). Isodtric dehydrogenase. The crude enzyme extract previously obtained was further fractionated with ammonium sulfate and ethanol according to the technique of Kornberg & Pricer (1951). The spectrophotometric assay of activity was that of Ochoa (1955) which follows the reduction of NADPH at 340 mtz using DLisocitrate as substrate. One unit of enzyme is that amount of enzyme which causes the reduction of ~M of NADPH in 20 min. Aconitase. The assay procedure of Racker (1950) using 0.33 M sodium citrate or 0.01 M sodium DL-isocitrate to form c~-aconitic acid was used. The spectrophotometric assay at 240 mlz is a measure of the number of unsaturated carbon linkages and has a number of limitations as pointed out by Racker (loc. dr.). One unit of activity has been defined as that amount of enzyme which will cause an initial rate of increase in absorbency at 240 mtz of 0.001/min. Fumarase. This enzyme was also determined by the methods of Racker (1950) except that the final assay mixture contained 0-05 M sodium 1-malate rather than the substrate used for aconitase determinations. Malate dehydrogenase. This enzyme was determined by the technique of Ochoa (1955). This technique was used without modifications and proved satisfactory as written. Protein determination. These were made following the technique of WaddeU (1956). Controls. Throughout the assay of these enzyme systems, control determinations were performed on colonal isolates (F~) of bakers' yeast. These preparations were grown aerobically in 2% glucose complex liquid media for 48 hr for study of
665
TCA CYCLE OF M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
TCA enzymes. It is well known that the levels of activity of the various enzymes can be manipulated according to the desires of the investigator by modifications in growth conditions. The authors have selected this colonal isolate to test the assay system employed and to provide some method of comparison of the activities observed in M . hirudinaceus with a known system. RESULTS AND DISCUSSION Table 1 presents information concerning four enzymes found in the TCA cycle. Of the four examined, M D H was found in high concentration and fumarase was found in small amounts. Aconitase and isocitric dehydrogenase were absent. Current information about this aspect of the metabolism of helminths is sketchy and incomplete. Reviewing the metabolism of cestodes, Read & Simmons (1963) TABLE 1 - - T R I C A R B O X Y L I C
Enzyme
Aconitase Isocitrate dehydrogenase with NADP+ Isocitrate dehydrogenase with NAD+ Fumarase Malate dehydrogenase
ACID CYCLE ENZYMES FROM FEMALE
1}I. hirudinaceus
Control Supematant of Mitochondrial pellet 34,800 g preparation 34,800 g preparation supernatant of 29,510 g preparation (activity/rag (activity/rag (activity/rag protein) protein) protein) 0"6 _+ 0"14
0"6 + 0"04
102"2
0"14+ 0"09
0-26+ 0"04
17"5
0"164- 0"09 2"0 _+ 0"3 1039 _+10
0"27+ 0"08 16"8 _+ 1"1 841 +_ 8
12-7 6"9 4"4
Specific activity based on units defined in Bergmeyer (1963) and mentioned in the Materials and Methods section. Controls were colonal isolates of bakers' yeast (F~.) grown aerobically for 48 hr prior to assay. summarized the evidence for an operative Krebs cycle in cestodes thus: "Evidence for oxidation of intermediates of the tricarboxylic acid cycle is incomplete" and "viewing all evidence available it must be concluded that the presence of a cycle has not been demonstrated". In an earlier review, Read (1961) had listed succinate, fumarate and malate as being metabolized but with negative findings with citrate, isocitrate and ~-ketoglutarate. However, this is not the case with nematodes in which the Krebs cycle has been demonstrated in some and not in others (Fairbairn, 1960). Oya et al. (1965) was unable to demonstrate the TCA cycle in Ascaris lumbricoides using enzyme assay techniques but did succeed in showing that such a system existed by using C 14 labelled intermediates. These authors concluded that although the TCA cycle existed that certain rate limiting steps made it of no practical importance to the parasite. It is also evident that a number of different helminths have what appears to be the last half of the Krebs cycle. As evidenced
666
T . T . D U N A O A N AND C. C. SCHEIFINGER
from Table I, M. hirudinaceusfallsinto the lattercategory. Of the four enzymes presented here, aconitase and isocitric dehydrogcnase received the majority of attention. At no time have we been encouraged to believe that these enzymes are present. Attempts to induce their formation by increasing the oxygen tcnsion (which works well with many single-cellfacultativeanaerobic organisms) of the medium in which they were maintained failed to give positive results. It is also interestingto note that compared to the controls the activityof fumarase is rather low whereas that of M D H is very high. There is no doubt that because of the absence of aconitase and isocitricdehydrogcnase M. hirudinaceusis predominately anaerobic in the type of metabolic systems employed. However, the high M D H activityand low fumarase activityare not readily explained. Bryant & Nicholas (1965), working with Moniliformis dubius, also failed to observe the first half of the Krebs cycle when its intermediates were sought; however, they did suggest that the TCA cycle was operative when there was an excess of one or more of the intermediates of the cycle. This was based on the presence of radioactive glutamate which occurred in small amounts following a long reaction time of 3 hr using a cell-free system. A large glutamate pool is understandable since many amino acids can be degraded to it. For this reason it is possible to find small amounts of radioactive glutamate following a long incubation. We, therefore, do not believe this is sufficient evidence for the presence of these reactions. There is no indication that ~-ketoglutarate and isocitrate were assayed by Bryant & Nicholas (1965). Since these are stable intermediates and are easy to isolate and measure, one would have expected some information on their presence or absence. Additional information may also have been obtained from a stepwise incubation. If the time of incubation was increased in increments of 10-15 min per mixture, an increase in products formed per increase in time should result. Had this been done, one could possibly tell the order in which the products are formed and obtain some idea of the sequence of events which takes place. This would uncover any radioactive compound occurring by the degradation of an intermediate formed earlier such as the formation of glutamate from the degradation of 1-ornithine, histidine, proline or arginine. From our own information we do not know if the enzyme substrates are present in the parasite. However, since we were unable to detect the enzymes by the assay systems employed, if the substrate is present the enzymes must not be inducible. We believe that the data of Bryant & Nicholas (1965) supports the enzyme data presented here. It would therefore appear that Acanthocephala do not possess a functional TCA system in the classical sense. REFERENCES BAHRG. F. & ZEITTERE. (1962) Study of mitochondria in rat liver. ~. Cell Biol. 15, 489-501. BEROMEYER HANS-ULRICH, Editor (1963) Methods of Enzymatic Analysis. Academic Press, New York. BRYANT C. & NICHOLASW. L. (1965) Intermediary metabolism in Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 15, 103-112.
TCA CYCLE OF M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
667
BUEDING E. & MOST H. (1953) Helminths: Metabolism, nutrition, and chemotherapy. A. Rev. Microbiol. 7, 295-326. FAIimAmN D. (1960) The physiology and biochemistry of Nematodes. In Nematology (Edited by SASSEI~J. N. & JENKINS W . R.). Univ. of North Carolina Press. KORNBEROA. & PRICERW. E. JR. (1951) Di- and triphosphopyridine nucleotide isocitric dehydrogenases in yeast, d~. b/ol. Chem. 189, 123-136. OCHOA S. (1955) Malic dehydrogenase from pig heart. In Methods in Enzymology, Vol. 1 (Edited by COLOWICKS. P. • KAPLANN. O.). Academic Press, New York. OYA H, KIK'dCHI G, BANDOT. & HAYASHIH. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides var. su/s. Expl Parasitol. 17, 229-240. RACKERE. (1950) Spectrophotometric measurement of the enzymatic formation of fumarase and ds-aconitic acids. Biochera. biophys. Acta 4, 211-214. READ C. P. (1961) The carbohydrate metabolism of worms. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTINA. W.). Univ. of Washington Press, Seattle, Washington. READ C. P. (1956) Carbohydrate metabolism of Hymenolepis diminuta. Expl Parasitol. 5, 325-344-. READC. P. & SIMMONSJ. E. JR. (1963) Biochemistry and physiology of tapeworms. Physiol. Rev. 43, 263-305. WADDELLW. J. (1956) A simple ultraviolet spectrophotometric method for the determination of protein..7. Lab. clin. Med. 48, 311-314.