Comp. Biochem. Physiol., 1975, VoL 50B, pp. 299 to 303. PergamonPress. Printed in Great Britain
ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN MACRACANTHORHYNCHUS HIRUDINACEUS (ACANTHOCEPHALA)* D. J. SAXONt AND T. T. DUNAGAN Department of Physiology, Southern Illinois University, Carbondale, Illinois 62901, U.S.A. (Received5February 1974)
Abstract--1. Transaldolase, 6-phosphogluconate dehydrogenase, transketolase, phosphoriboisomerase and D-xylulose-5-phosphate-3-epimeras¢ were studied in extracts of Macracanthorhynehus hirudinaceus. 2. Enzymes of the pentose phosphate pathway were observed throughout M. hirudinaceus and no specific site indicated for this pathway by analyzing activities in the anterior third, which contains the praesoma and lerrmisci; middle third, containing pseudocoelomate fluid and body wall musculature; and posterior third, containing reproductive organs. 3. No significant difference was observed in the specific activity of the enzymes from separate sexes.
INTRODUCTION THE CARBOHYDRATEmetabolism of Acanthocephala has been studied by a number of investigators. Unfortunately, as indicated by Fig. 1, there are several steps in the pathway for which no data have been collected and those which are available generally represents only a few of the larger species rather than a good representation of the three major taxonomic divisions of the Acanthocephala. Until now there has been no extensive study of the pentose phosphate pathway in this phylum. Dunagan & Scheifinger (1966b) reported a high specific activity of glucose-6phosphate dehydrogenase (G6PDH) in Macracanthorhynchus hirudinaceus which was later ioslated and characterized by Stein (1971). Kbrting & Fairbain (1972) and Horvath (1972) found many glycolytic enzymes in Moniliformis dubious including glucose-6phosphate dehydrogenase and phosphoguconate dehydrogenase (6PGDH). Some enzymes of the pentose phosphate pathway have been studied in other helminths. De Ley & Vercruysse (1955) noted G 6 P D H and 6 P G D H in a large number of cestodes and nematodes. Waitz (1963) found G 6 P D H in Hydatigera taeniaeformis. Read & Simons (1963) reported that homogenates of Hymenolepis diminuta formed sedoheptulose-7phosphate and ribose-5-phosphate. Agosin and * This investigation is a part of the senior author's dissertation for the Ph.D. degree at Southern Illinois University. Financial assistance was received from the Office of Research and Projects, Southern Illinois University. t Present address: Department of Biological Sciences, Morehead State University, Morehead, Kentucky 40351, U.S.A.
Aravena (1960) noted the occurrence of this pathway in Echinococcus granulosus. More specifically, Entner (1957) found enzymes of this pathway, including transketolase (TK), in Ascaris lumbricoides var suum. Transaldolase (TA) was not assayed. Langer et aL (1971) reported pentose phosphate pathway enzymes in A. suum. In this latter study phosphoriboisomerase 0 ) and Dxylulose-5-phosphate-3-epimerase (E) were measured jointly, and T A was noted. Srivastava et aL (1970) found 6 P G D H in Ascaridia galli. It is the purpose of this study to provide information concerning the presence of 6PGDH, TA, E, I and TK, indicating an intact pentose phosphate pathway in M. hirudinaceus. This study presents comparative data on the specific activities of pentose phosphate pathway enzymes from different body divisions of male and female M. hirudinaceus. MATERIALS AND METHODS Chemicals
The analytical grade reagents and enzymes except TA were purchased from Sigma Chemical Company, Saint Louis, Missouri. Water, twice distilled in glass, was used in all preparations. It should be noted that the erythrose4-phosphate (E-4-P) solution is very unstable and was prepared in small aliquots on the day used and kept at 15°C until introduction into the reaction mixture. Transaldolase was a gift from Dr. B. L. Horecker of Yeshiva University, Bronx, New York. Enzyme solubilization
299
The worms were collected from the small intestine of slaughtered swine at Hunter Packing Company, East
300
D.J. SAXONAND T. T. DUNAGAN 8,3
UDPG
Glycogen I
Trehalose ]
V Glucose-l-phosphate
Maltose~ 67[ 8'3 Glucose
5
ivL Glucose-6-Phosphate
Turanose ._~6 Fructose
~,
:~. 6-Phosphogluconate COz'l"-~ 11, 14 14 Ribulose-5-phosphate
Fructose-6-Phosphate Pentose phosphate pathway
Fructose-l,6-diphosphate [ 10, 13 13 Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate
10 ,
1,3 Diphosphoglycerate I I
3-Phosphoglycerate I 1
~,. 2-Phosphoglycerate
,10
phospho-enol-pyruvate
Ethanol
~1l ~0,~ Pyruvate ~
CO~---~ 9
co2+-
--
Lactate
T 11 Alanine ,i~
Acetaldehyde
1
11
Acetate
Acetyl-CoA I
/ ~
Citrate ,,
q
Oxaloa :etate ~'4 [--~ M~a~e ~
/ Glyoxylate ~
I Fumarate
co~-~] 9
~1, 2 Succinate • ~ _
Pyruvate
// /
]~
Cis-aconitate ,~, Isocitrate Oxalosuccinate
'--~co
i1~/ alpha-Ketoglutarate ~ - - Succinyl-CoA I k ' ~ - ~ - - ~ ' C O 2 ~ 5 Glutamate
Fig. 1. A composite summary of the carbohydrate metabolism in Acanthocephala is illustrated. Unestablished reactions ( . . . . . . ~) are indicated. Established reactions ( > ) are referenced with numerals. The references are as follows: 1. Bryant & Nicholas (1965). 8. Graft (1964). 2. Bryant & Nicholas (1966). 9. Horvath (1972). 3. Crompton & Lockwood (1968). 10. Horvath & Fisher (1971). 4. Dunagan & Scheifinger (1966a). 11. KiSrting & Fairbairn (1972). 5. Dunagan & Scheifinger (1966b). 12. McAlister & Fisher (1972). 6. Dunagan & Yau (1968). 13. Read (1961). 7. Fisher (1964). 14. Saxon & Dunagan (1974).
Enzymes of the pentose phosphate pathway in Macracanthorhynchus Saint Louis, Illinois. They were placed in Dewar flasks, pre-warmed, containing sufficient swine small intestinal contents to cover the specimens. After collection, all adhering material was removed from the proboscis and they were washed in cold tyrodes solution to remove residual intestinal juice. Following washing the worms were blotted dry and weighed. The remaining process of homogenate preparation was performed at approximately 10°C. The specimens were minced before being homogenized in a cold mortar and pestle containing 1 g of silicon and 2 ml of appropriate buffer per g of sample tissue. The buffer utilized depended upon the enzyme to be assayed. Homogenates of whole worms, pseudocoelomic fluid and body wall of the anterior, middle and posterior one-thirds of males and females were centrifuged at 0°C for 30 min at 20,000 g and the resulting supernatant used for the enzyme assays. Enzyme assay
One unit of enzyme is defined as the amount of enzyme which converts 1 t~mole of substrate per min. The specific activity is the number of enzyme units per mg of protein. Protein was determined by the method of Lowry et al. (1951). Standard curves from bovine serum albumin were always repeated with each experimental analysis. An enzyme extract dilution of 1/199 was used for protein determination. The respective enzyme activities were measured at 25°C by determining either the rate of reduction of NAD + or NADP + or oxidation of NADH. These measurements were made at 340 nm with a Beckman Model DB spectrophotometer in combination with a 10-in. potentiometric recorder. Temperature was controlled in the cuvette chamber by a thermocirculator with a low temperature accessory. Standard reactions with enzymes of known specific activity were performed with each group of reagents to ensure maximal activity. A control consisting of all the reaction mixture except extract was utilized. Additional controls using reaction mixture minus substrates and/or indicator enzymes were also used. Controls of standard reactions with additions of enzyme extract were utilized to test for possible inhibition of the standard reaction by some possible contaminant in the extract. Each analysis was performed a minimum of three times. The determination of 6PGDH was essentially that of de Ley & Vercruysse (1955). Transaldolase was determined according to the method of Racker (1963a). A control of reaction mixture minus E-4-P was used to check for interfering phosphoglucoisomerase activity. The method of Racker (1963c) was used for the TK assay. The procedure used to determine I was essentially that of Racker (1963b). In this series of coupled reactions a time lag of greater than 5 rain occurred before a stable kinetic rate was reached. Twofold increases in the auxiliary and indicator enzymes did not reduce the time lag. A similar time lag was noted by Langer et aL (1971). The determination of E in extracts is a problem since ribulose-5-phosphate (Ru-5-P) can either be epimerized to xylulose-5-phosphate (Xu-5-P) or isomerized to ribose-5phosphate (R-5-P). Based on a modification of the general procedure presented by Racker (1962) excessive
301
R-5-P in the presence of standard I enables the Ru-5-P generated to be converted to Xu-5-P by the E in the extract. RESULTS AND DISCUSSION This study indicates the presence of 6-phosphogluconate dehydrogenase, transaldolase, transketolase, phosphoriboisomerase and o-xylulose-5-phosphate-3-epimerase in extracts of male and female M. hirudinaceus. There were no appreciable differences in the enzymes in either sex (Table 1). This is the first study in which these enzymes have been reported in Acanthocephala. Only the studies of Entner (1957), Agosin & Arevena (1960), and Langer et al. (1971) presented data on as many pentose phosphate enzymes from helminths. The enzyme with the greatest activity in both male and female worms was 6-phosphogluconate dehydrogenase. The limiting activity in females was the epimerase, and in males the isomerase and epimerase (Table 1). Table 1. Pentose phosphate pathway enzymes in male and female M. hirudinaeeus Specific activity* Enzyme 6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphoriboisomerase Xylulose-5-phosphate-3-epimerase
Male
Female
0"0258 0"0108 0.0099 0.0075 0.0080
0.0204 0.0110 0.0087 0"0080 0.0065
* Specific activity is expressed as p.moles of substrate utilized per min per mg of protein (25°C). The posterior portion of M . hirudinaceus has been suggested by Stein (1971) as the possible site of the pentose phosphate pathway based on his observation that glucose-6-phosphate dehydrogenase activity was highest in that part of the worm. Of the different body divisions analyzed in this study there was no one major site found for the pentose phosphate pathway (Table 2). The enzyme with the greatest activity, in both sexes, was 6-phosphogluconate dehydrogenase which was highest in the middle division of the helminth rather than the posterior most division. The reasons for the differences between these observations and those of Stein (1971) are unclear at this time. The lowest activity in females was xylulose-5-phosphate-3-epimerase in the middle division. Transaldolase and transketolase exhibited the lowest enzyme activities in the anterior division of males. One specific site for the pentose phosphate cycle is not indicated by these results. Pseudocoelomic fluid was analyzed for these enzymes and transketolase activity was not detected in either sex (Table 3). The highest activity was 6phosphogluconate dehydrogenase in the anterior
302
D. J. SAXONAND T. T. DUNAGAN Table 2.
Pentose phosphate pathway enzymes of divisions from male and female M. hirudinaceus Specific activity* Anterior third Enzyme
6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphroiboisomerase Xylulose-5-phosphate-3epimerase
Middle third
Posterior third
Male
Female
Male
Female
Male
Female
0.0133
0.0115
0.0180
0.0182
0.0147
0.0157
0.0040 0.0039 0.0061 0.0047
0.0059 0.0060 0.0040 0.0034
0.0048 0.0052 0"0061 0-0043
0.0061 0.0050 0-0046 0.0026
0.0040 0.0051 0.0054 0.0052
0-0046 0.0050 0.0037 0.0037
* Specific activity expressed as/,moles of substrate utilized per min per mg protein (25°C). Table 3.
Pentose pathway enzymes of pseudocoelomic fluids in M. hirudinaceus Specific activity* Anterior third
Enzyme 6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphoriboisomerase Xylulose-5-phosphate-3 epimerase
Middle third
Posterior third
Male
Female
Male
Female
Male
Female
0.0121
0.0093
0.0093
0.0066
0-0079
0.0108
0.0049 0.0000 0.0067 0.0067
0.0049 0.0000 0.0046 0.0047
0.0046 0.0000 0.0066 0.0057
0.0052 0.0000 0-0052 0-0052
0.0056 0.0000 0-0082 0.0061
0.0059 0.0000 0.0048 0.0055
* Specific activity expressed as tzmoles of substrate utilized per min per mg protein (25°C). pseudocoelomic fluid of males and in the posterior pseudocoelomic fluid of females. Since 6-phosphogluconate dehydrogenase, transaldolase, transketolase, phosphoriboisomerase and o-xylulose-5-phosphate-3-epimerase are present in male and female M. hirudinaceus, this indicates an intact pentose phosphate pathway is present. H o w ever, the extent to which this pathway is operable in 34. hirudinaceus has not been determined. REFERENCES AGOSIN M. & ARAVENAL. (1960) Studies on the metabolism of Echinococcus granulosus.--IV Enzymes of the pentose phosphate pathway. Expl Paras#. 10, 28-38. BRYANTC. & NICHOLASW. L. (1965) Intermediary metabolism in Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 15, 103-112. BRYANT C. & NICHOLAS W. L. (1966) Studies on the oxidative metabolism of Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 17, 825-840. CROMPTON D. W. T. & LOCKWOOD A. P. M. (1968) Studies on the absorption and metabolism of D(U-14 C) glucose by Polymorphus minutus (Acanthoeephala) in vitro. J. exp. Biol. 48, 411-425. DUNAGAN T. T. • SCHEIFINGERC. C. (1966a) Studies on the TCA cycle of Macracanthorhynchus hirudinaceus (Acanthocephala). Comp. Biochem. PhysioL 18, 663667.
DUNAGANT. T. & SCHE1FINGERC. C. (1966b) Studies on glycolytic enzymes from Macracanthorhynchus hirudinaceus (Acanthocephala). J. Parasitol. 52, 730-734. DUNAGANT. T. & YAU T. M. (1968) Oligosaccharidases from Macracanthorhynchus hirudinaceus (Acanthocephala) from swine. Comp. Biochem. Physiol. 26, 281-289. ENTNER N. (1957) The occurrence of the pentose phosphate pathway in Ascaris lumbricoides. Arch Biochem. Biophys. 71, 52-61. FISHER F. M. (1964) Synthesis of trehalose in Acanthocephala. J. Parasitol. 50, 803-804. GRAFF D. J. (1964) Metabolism of C14-glucose by Moniliformis dubhls (Acanthocephala). J. Parasitol. 50, 230-234. HORVATH K. (1972) Glycolytic enzymes in larval Moniliformis dubius. J. Parasitol. 58, 1219-1220. HORVATH K. & FISHER M., JR. (1971) Enzymes of CO2 fixation in larval and adult Moniliformis dubius (Acanthocephala). J. Parasitol. 57, 440-442. KORTINGW. & FAIRBAINO. (1972) Anaerobic metabolism in Moniliformis dubius (Acanthocephala). J. Parasitol. 58, 45-50. LANGER B. W., JR., SMITrt W. J. & THEODORIDESV. J. (1971) The pentose cycle in adult Ascaris suum. J. Parasitol. 57, 485-486. DE LEY J. & VERCRUVSSER. (1955) Glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase in worms. Biochem. biophys. Acta 16, 615-616. LOWRY, O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
Enzymes of the pentose phosphate pathway in Macracanthorhynchus MCALISTERR. O. & FtsrmR F. M. (1972) The biosynthesis of trehalose in Moniliformis dubius (Acanthocephala). J. Parsitol. 58, 51-62. RACKER E. (1962) D-Xylulose-5-phosphate-3-epimerase from muscle. In Methods of Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.), Vol. V, pp. 280282. Academic Press, New York. RACKER E. (1963a) D-Erythrose-4-phosphate. In Methods of Enzymatic Analysis (Edited by BERt3MEYER, H. U.), pp. 205-207. Academic Press, New York. RACKER E. (1963b) D-Ribose-5-phosphate. In Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.), pp. 175-177. Academic Press, New York. RACKERE. (1963c) D-Xylulose-5-phosphate. In Methods of Enzymatic Analysis (Edited by BEROMEYERH. U.), pp. 201-204. Academic Press, New York. READ C. P. (1961) The carbohydrate metabolism of worms. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTIN A. W.), pp. 3-34. University of Washington Press, Seattle.
303
READ C. P. & SIMMONSJ. E., JR. (1963) Biochemistry and physiology of tapeworm. Physiol. Rev. 43, 263-305. SRIVASTAVAV. M. L., GrlATAKS. & MURTI C. R. K. (1970) Ascaridia galli: lactic acid production-glycogen content, glycolytic enzymes, and properties of purified aldolase, enolase and glucose-6-phosphate dehydrogenase. Parasit. 60, 157-180. STEINT. A. (1971) Purification and characterization of Dglucose-6-phosphate dehydrogenase in Macracanthorhynchus hirudinaceus. Comp. Biochem. Physiol. 39, 541-549. WArrz J. A. (1963) Glycolytic enzymes of the cestode Hydatigera taeniaeformis. J. ParasitoL 49, 285-293.
Key Word Index--Acanthocephala; Macracanthorhynchus hirudinaceus; pentose phosphate shunt; transaldolase; 6-phosphogluconate dehydrogenase; transketolase; phosphoriboisomerase; D-xylulose-5-phosphate3- epimerase.
ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN MACRACANTHORHYNCHUS HIRUDINACEUS (ACANTHOCEPHALA)* D. J. SAXONt AND T. T. DUNAGAN Department of Physiology, Southern Illinois University, Carbondale, Illinois 62901, U.S.A. (Received5February 1974)
Abstract--1. Transaldolase, 6-phosphogluconate dehydrogenase, transketolase, phosphoriboisomerase and D-xylulose-5-phosphate-3-epimeras¢ were studied in extracts of Macracanthorhynehus hirudinaceus. 2. Enzymes of the pentose phosphate pathway were observed throughout M. hirudinaceus and no specific site indicated for this pathway by analyzing activities in the anterior third, which contains the praesoma and lerrmisci; middle third, containing pseudocoelomate fluid and body wall musculature; and posterior third, containing reproductive organs. 3. No significant difference was observed in the specific activity of the enzymes from separate sexes.
INTRODUCTION THE CARBOHYDRATEmetabolism of Acanthocephala has been studied by a number of investigators. Unfortunately, as indicated by Fig. 1, there are several steps in the pathway for which no data have been collected and those which are available generally represents only a few of the larger species rather than a good representation of the three major taxonomic divisions of the Acanthocephala. Until now there has been no extensive study of the pentose phosphate pathway in this phylum. Dunagan & Scheifinger (1966b) reported a high specific activity of glucose-6phosphate dehydrogenase (G6PDH) in Macracanthorhynchus hirudinaceus which was later ioslated and characterized by Stein (1971). Kbrting & Fairbain (1972) and Horvath (1972) found many glycolytic enzymes in Moniliformis dubious including glucose-6phosphate dehydrogenase and phosphoguconate dehydrogenase (6PGDH). Some enzymes of the pentose phosphate pathway have been studied in other helminths. De Ley & Vercruysse (1955) noted G 6 P D H and 6 P G D H in a large number of cestodes and nematodes. Waitz (1963) found G 6 P D H in Hydatigera taeniaeformis. Read & Simons (1963) reported that homogenates of Hymenolepis diminuta formed sedoheptulose-7phosphate and ribose-5-phosphate. Agosin and * This investigation is a part of the senior author's dissertation for the Ph.D. degree at Southern Illinois University. Financial assistance was received from the Office of Research and Projects, Southern Illinois University. t Present address: Department of Biological Sciences, Morehead State University, Morehead, Kentucky 40351, U.S.A.
Aravena (1960) noted the occurrence of this pathway in Echinococcus granulosus. More specifically, Entner (1957) found enzymes of this pathway, including transketolase (TK), in Ascaris lumbricoides var suum. Transaldolase (TA) was not assayed. Langer et aL (1971) reported pentose phosphate pathway enzymes in A. suum. In this latter study phosphoriboisomerase 0 ) and Dxylulose-5-phosphate-3-epimerase (E) were measured jointly, and T A was noted. Srivastava et aL (1970) found 6 P G D H in Ascaridia galli. It is the purpose of this study to provide information concerning the presence of 6PGDH, TA, E, I and TK, indicating an intact pentose phosphate pathway in M. hirudinaceus. This study presents comparative data on the specific activities of pentose phosphate pathway enzymes from different body divisions of male and female M. hirudinaceus. MATERIALS AND METHODS Chemicals
The analytical grade reagents and enzymes except TA were purchased from Sigma Chemical Company, Saint Louis, Missouri. Water, twice distilled in glass, was used in all preparations. It should be noted that the erythrose4-phosphate (E-4-P) solution is very unstable and was prepared in small aliquots on the day used and kept at 15°C until introduction into the reaction mixture. Transaldolase was a gift from Dr. B. L. Horecker of Yeshiva University, Bronx, New York. Enzyme solubilization
299
The worms were collected from the small intestine of slaughtered swine at Hunter Packing Company, East
300
D.J. SAXONAND T. T. DUNAGAN 8,3
UDPG
Glycogen I
Trehalose ]
V Glucose-l-phosphate
Maltose~ 67[ 8'3 Glucose
5
ivL Glucose-6-Phosphate
Turanose ._~6 Fructose
~,
:~. 6-Phosphogluconate COz'l"-~ 11, 14 14 Ribulose-5-phosphate
Fructose-6-Phosphate Pentose phosphate pathway
Fructose-l,6-diphosphate [ 10, 13 13 Glyceraldehyde-3-phosphate Dihydroxyacetone phosphate
10 ,
1,3 Diphosphoglycerate I I
3-Phosphoglycerate I 1
~,. 2-Phosphoglycerate
,10
phospho-enol-pyruvate
Ethanol
~1l ~0,~ Pyruvate ~
CO~---~ 9
co2+-
--
Lactate
T 11 Alanine ,i~
Acetaldehyde
1
11
Acetate
Acetyl-CoA I
/ ~
Citrate ,,
q
Oxaloa :etate ~'4 [--~ M~a~e ~
/ Glyoxylate ~
I Fumarate
co~-~] 9
~1, 2 Succinate • ~ _
Pyruvate
// /
]~
Cis-aconitate ,~, Isocitrate Oxalosuccinate
'--~co
i1~/ alpha-Ketoglutarate ~ - - Succinyl-CoA I k ' ~ - ~ - - ~ ' C O 2 ~ 5 Glutamate
Fig. 1. A composite summary of the carbohydrate metabolism in Acanthocephala is illustrated. Unestablished reactions ( . . . . . . ~) are indicated. Established reactions ( > ) are referenced with numerals. The references are as follows: 1. Bryant & Nicholas (1965). 8. Graft (1964). 2. Bryant & Nicholas (1966). 9. Horvath (1972). 3. Crompton & Lockwood (1968). 10. Horvath & Fisher (1971). 4. Dunagan & Scheifinger (1966a). 11. KiSrting & Fairbairn (1972). 5. Dunagan & Scheifinger (1966b). 12. McAlister & Fisher (1972). 6. Dunagan & Yau (1968). 13. Read (1961). 7. Fisher (1964). 14. Saxon & Dunagan (1974).
Enzymes of the pentose phosphate pathway in Macracanthorhynchus Saint Louis, Illinois. They were placed in Dewar flasks, pre-warmed, containing sufficient swine small intestinal contents to cover the specimens. After collection, all adhering material was removed from the proboscis and they were washed in cold tyrodes solution to remove residual intestinal juice. Following washing the worms were blotted dry and weighed. The remaining process of homogenate preparation was performed at approximately 10°C. The specimens were minced before being homogenized in a cold mortar and pestle containing 1 g of silicon and 2 ml of appropriate buffer per g of sample tissue. The buffer utilized depended upon the enzyme to be assayed. Homogenates of whole worms, pseudocoelomic fluid and body wall of the anterior, middle and posterior one-thirds of males and females were centrifuged at 0°C for 30 min at 20,000 g and the resulting supernatant used for the enzyme assays. Enzyme assay
One unit of enzyme is defined as the amount of enzyme which converts 1 t~mole of substrate per min. The specific activity is the number of enzyme units per mg of protein. Protein was determined by the method of Lowry et al. (1951). Standard curves from bovine serum albumin were always repeated with each experimental analysis. An enzyme extract dilution of 1/199 was used for protein determination. The respective enzyme activities were measured at 25°C by determining either the rate of reduction of NAD + or NADP + or oxidation of NADH. These measurements were made at 340 nm with a Beckman Model DB spectrophotometer in combination with a 10-in. potentiometric recorder. Temperature was controlled in the cuvette chamber by a thermocirculator with a low temperature accessory. Standard reactions with enzymes of known specific activity were performed with each group of reagents to ensure maximal activity. A control consisting of all the reaction mixture except extract was utilized. Additional controls using reaction mixture minus substrates and/or indicator enzymes were also used. Controls of standard reactions with additions of enzyme extract were utilized to test for possible inhibition of the standard reaction by some possible contaminant in the extract. Each analysis was performed a minimum of three times. The determination of 6PGDH was essentially that of de Ley & Vercruysse (1955). Transaldolase was determined according to the method of Racker (1963a). A control of reaction mixture minus E-4-P was used to check for interfering phosphoglucoisomerase activity. The method of Racker (1963c) was used for the TK assay. The procedure used to determine I was essentially that of Racker (1963b). In this series of coupled reactions a time lag of greater than 5 rain occurred before a stable kinetic rate was reached. Twofold increases in the auxiliary and indicator enzymes did not reduce the time lag. A similar time lag was noted by Langer et aL (1971). The determination of E in extracts is a problem since ribulose-5-phosphate (Ru-5-P) can either be epimerized to xylulose-5-phosphate (Xu-5-P) or isomerized to ribose-5phosphate (R-5-P). Based on a modification of the general procedure presented by Racker (1962) excessive
301
R-5-P in the presence of standard I enables the Ru-5-P generated to be converted to Xu-5-P by the E in the extract. RESULTS AND DISCUSSION This study indicates the presence of 6-phosphogluconate dehydrogenase, transaldolase, transketolase, phosphoriboisomerase and o-xylulose-5-phosphate-3-epimerase in extracts of male and female M. hirudinaceus. There were no appreciable differences in the enzymes in either sex (Table 1). This is the first study in which these enzymes have been reported in Acanthocephala. Only the studies of Entner (1957), Agosin & Arevena (1960), and Langer et al. (1971) presented data on as many pentose phosphate enzymes from helminths. The enzyme with the greatest activity in both male and female worms was 6-phosphogluconate dehydrogenase. The limiting activity in females was the epimerase, and in males the isomerase and epimerase (Table 1). Table 1. Pentose phosphate pathway enzymes in male and female M. hirudinaeeus Specific activity* Enzyme 6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphoriboisomerase Xylulose-5-phosphate-3-epimerase
Male
Female
0"0258 0"0108 0.0099 0.0075 0.0080
0.0204 0.0110 0.0087 0"0080 0.0065
* Specific activity is expressed as p.moles of substrate utilized per min per mg of protein (25°C). The posterior portion of M . hirudinaceus has been suggested by Stein (1971) as the possible site of the pentose phosphate pathway based on his observation that glucose-6-phosphate dehydrogenase activity was highest in that part of the worm. Of the different body divisions analyzed in this study there was no one major site found for the pentose phosphate pathway (Table 2). The enzyme with the greatest activity, in both sexes, was 6-phosphogluconate dehydrogenase which was highest in the middle division of the helminth rather than the posterior most division. The reasons for the differences between these observations and those of Stein (1971) are unclear at this time. The lowest activity in females was xylulose-5-phosphate-3-epimerase in the middle division. Transaldolase and transketolase exhibited the lowest enzyme activities in the anterior division of males. One specific site for the pentose phosphate cycle is not indicated by these results. Pseudocoelomic fluid was analyzed for these enzymes and transketolase activity was not detected in either sex (Table 3). The highest activity was 6phosphogluconate dehydrogenase in the anterior
302
D. J. SAXONAND T. T. DUNAGAN Table 2.
Pentose phosphate pathway enzymes of divisions from male and female M. hirudinaceus Specific activity* Anterior third Enzyme
6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphroiboisomerase Xylulose-5-phosphate-3epimerase
Middle third
Posterior third
Male
Female
Male
Female
Male
Female
0.0133
0.0115
0.0180
0.0182
0.0147
0.0157
0.0040 0.0039 0.0061 0.0047
0.0059 0.0060 0.0040 0.0034
0.0048 0.0052 0"0061 0-0043
0.0061 0.0050 0-0046 0.0026
0.0040 0.0051 0.0054 0.0052
0-0046 0.0050 0.0037 0.0037
* Specific activity expressed as/,moles of substrate utilized per min per mg protein (25°C). Table 3.
Pentose pathway enzymes of pseudocoelomic fluids in M. hirudinaceus Specific activity* Anterior third
Enzyme 6-Phosphogluconate dehydrogenase Transaldolase Transketolase Phosphoriboisomerase Xylulose-5-phosphate-3 epimerase
Middle third
Posterior third
Male
Female
Male
Female
Male
Female
0.0121
0.0093
0.0093
0.0066
0-0079
0.0108
0.0049 0.0000 0.0067 0.0067
0.0049 0.0000 0.0046 0.0047
0.0046 0.0000 0.0066 0.0057
0.0052 0.0000 0-0052 0-0052
0.0056 0.0000 0-0082 0.0061
0.0059 0.0000 0.0048 0.0055
* Specific activity expressed as tzmoles of substrate utilized per min per mg protein (25°C). pseudocoelomic fluid of males and in the posterior pseudocoelomic fluid of females. Since 6-phosphogluconate dehydrogenase, transaldolase, transketolase, phosphoriboisomerase and o-xylulose-5-phosphate-3-epimerase are present in male and female M. hirudinaceus, this indicates an intact pentose phosphate pathway is present. H o w ever, the extent to which this pathway is operable in 34. hirudinaceus has not been determined. REFERENCES AGOSIN M. & ARAVENAL. (1960) Studies on the metabolism of Echinococcus granulosus.--IV Enzymes of the pentose phosphate pathway. Expl Paras#. 10, 28-38. BRYANTC. & NICHOLASW. L. (1965) Intermediary metabolism in Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 15, 103-112. BRYANT C. & NICHOLAS W. L. (1966) Studies on the oxidative metabolism of Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 17, 825-840. CROMPTON D. W. T. & LOCKWOOD A. P. M. (1968) Studies on the absorption and metabolism of D(U-14 C) glucose by Polymorphus minutus (Acanthoeephala) in vitro. J. exp. Biol. 48, 411-425. DUNAGAN T. T. • SCHEIFINGERC. C. (1966a) Studies on the TCA cycle of Macracanthorhynchus hirudinaceus (Acanthocephala). Comp. Biochem. PhysioL 18, 663667.
DUNAGANT. T. & SCHE1FINGERC. C. (1966b) Studies on glycolytic enzymes from Macracanthorhynchus hirudinaceus (Acanthocephala). J. Parasitol. 52, 730-734. DUNAGANT. T. & YAU T. M. (1968) Oligosaccharidases from Macracanthorhynchus hirudinaceus (Acanthocephala) from swine. Comp. Biochem. Physiol. 26, 281-289. ENTNER N. (1957) The occurrence of the pentose phosphate pathway in Ascaris lumbricoides. Arch Biochem. Biophys. 71, 52-61. FISHER F. M. (1964) Synthesis of trehalose in Acanthocephala. J. Parasitol. 50, 803-804. GRAFF D. J. (1964) Metabolism of C14-glucose by Moniliformis dubhls (Acanthocephala). J. Parasitol. 50, 230-234. HORVATH K. (1972) Glycolytic enzymes in larval Moniliformis dubius. J. Parasitol. 58, 1219-1220. HORVATH K. & FISHER M., JR. (1971) Enzymes of CO2 fixation in larval and adult Moniliformis dubius (Acanthocephala). J. Parasitol. 57, 440-442. KORTINGW. & FAIRBAINO. (1972) Anaerobic metabolism in Moniliformis dubius (Acanthocephala). J. Parasitol. 58, 45-50. LANGER B. W., JR., SMITrt W. J. & THEODORIDESV. J. (1971) The pentose cycle in adult Ascaris suum. J. Parasitol. 57, 485-486. DE LEY J. & VERCRUVSSER. (1955) Glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase in worms. Biochem. biophys. Acta 16, 615-616. LOWRY, O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
Enzymes of the pentose phosphate pathway in Macracanthorhynchus MCALISTERR. O. & FtsrmR F. M. (1972) The biosynthesis of trehalose in Moniliformis dubius (Acanthocephala). J. Parsitol. 58, 51-62. RACKER E. (1962) D-Xylulose-5-phosphate-3-epimerase from muscle. In Methods of Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.), Vol. V, pp. 280282. Academic Press, New York. RACKER E. (1963a) D-Erythrose-4-phosphate. In Methods of Enzymatic Analysis (Edited by BERt3MEYER, H. U.), pp. 205-207. Academic Press, New York. RACKER E. (1963b) D-Ribose-5-phosphate. In Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.), pp. 175-177. Academic Press, New York. RACKERE. (1963c) D-Xylulose-5-phosphate. In Methods of Enzymatic Analysis (Edited by BEROMEYERH. U.), pp. 201-204. Academic Press, New York. READ C. P. (1961) The carbohydrate metabolism of worms. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTIN A. W.), pp. 3-34. University of Washington Press, Seattle.
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Key Word Index--Acanthocephala; Macracanthorhynchus hirudinaceus; pentose phosphate shunt; transaldolase; 6-phosphogluconate dehydrogenase; transketolase; phosphoriboisomerase; D-xylulose-5-phosphate3- epimerase.
