EXPERIMENTAL
41,89-94
PARASITOLOGY
fascia/a hepatica: Electrophoretic
( 1977)
The Subcellular Distribution and Kinetic Properties of Malate Dehydrogenase A. J.
Department
of Applied
(Accepted
PROBERT
AND
T. LWIN 1
Zoology, Bangor,
University Gwynedd,
College U.K.
for
publication
2 March
of North
and
Wales,
1976)
PROBEI~T, A. J., AND LWIS, T. 1977. Fasciola Izepatica: The subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase. Experimental Parasitology 41, 89-94. The optimal pH for oxalacetate reduction was 9.0 for the mitochondrial and supernatant enzyme. The optimal pH for malate oxidation was 10 for both fractions. Maximal activity during oxalacetate reduction was 3.26 * 0.26 pmoles of NADH oxidized/ mg of protein/min and 3.02 * 0.27 for the supernatant and mitochondrial fractions, respectively. Maximal malate oxidation was 0.495 f 0.08 and 0.72 f 0.12 (not statistically different) for the supernatant and mitochondrial fractions, respectively. High concentrations of malate inhibited activity to a greater extent in the supernatant fraction while oxalacetate inhibited activity to a greater extent in the mitochondrial fraction. The supematant malate dehydrogenase was more heat stable than the mitochrondrial enzyme. Electrophoresis showed three isoenzymes of malate dehydrogenase in whole homogenates of Fascioka hepatica and all three were represented in the supernatant while only one band was seen in the mitochondrial fraction. The presence of two types of malate dehydrogenase, one in the supematant with three isoenzymes and one in the mitochondria with one isoenzyme, is proposed. INDEX
DESCRIPTORS:
and EC 1.1.1.39); geneity; Subcellular
FascioZu hepatica; Trematode; Malate dehydrogenase Lactate dehydrogenase (EC 1.1.1.27); Biochemistry; distribution; Electrophoretic properties.
INTRODUCTION
Pennoit-De Cooman and van Grembergen (1942) first reported the presence of malate dehydrogenase in Fasciola hepatica and this was confirmed later by Prichard and Schofield (1968a). It has been postulated by Prichard and Schofield (1968b) and De Zoeten et al. (1969) that malate dehydrogenase catalyzes the reduction of oxalacetate to a greater extent than malate oxidation in F. hepatica. This leads to the production
of
1 Present Parasitology, Veterinary
Copyright All rights
succinnte,
which
is coupled
address: Department of Veterinary Institute of Animal Husbandry and Science, Rangoon, Burma.
6 1977 by Academic of reproduction in any
Press, Inc. form reserved.
(EC Enzyme
1.1.1.37 hetero-
with ATP synthesis, thereby generating energy by reversal of some steps in the tricarboxylic acid cycle, rather than by oxidative processes. However, evidence for the presence of all the enzymes and intcrmediates necessary for a functional TCA cycle in F. hepatica has been reported (Thorsell 1963; Prichard and Schofield 1968a; De Zoeten et al. 1969). In view of the important position which malate dehydrogenase occupies in the metabolism of F. hepatica, studies have been undertaken on the subcellular distribution and the kinetic and electrophoretic properties of this enzyme.
ISSN
0014-4894
90
PROBERT AND LWIN MATFJUALSANDMETHODS
Preparation of homogenates. Ten percent (w/v) homogenates of adult flukes from cattle were prepared according to the method of Probert and Lwin (1974). Mitochondrial and supematant fractions were prepared by the method of Prichard and Schofield ( 1968a). The protein concentration was measured in each preparation by the biuret method (Kabat and Mayer 1961). Preparations were diluted with 0.25 M sucrose to given the desired concentrations. Enzyme assays. Assays were conducted spectrophotometrically using a Cecil ultraviolet spectrophotometer using thermoregulated cuvettes and on external pen recorder. All assayswere carried out at 25 C in a final volume of 3 ml and, except where otherwise stated, the reaction mixture was preincubated at the experimental temperature for 5 min prior to the addition of the substrate. Malate dehydrogenase (EC 1.1.1.37) was measured by the method of Shonk and Boxer ( 1964). The assaymedium contained triethanolamine plus EDTA buffer, 0.05 M; NADH, 0.234 mM; homogenate, 0.05 mg of protein; and cis-oxalacetic acid (neutralized), 0.33 mM. The oxidation of malate was assayed by the method of Prichard and Schofield ( 1968a). The medium contained glycine-NaOH buffer, 0.1 M; NAD, 1 mM; homogenate, 0.1 mg of protein; and L-malic acid (neutralized), 10 mM. The reaction in each case was started by addition of substrate and activity recorded from the increase or decrease in extinction at 340 nm. Malate dehydrogenase ( decarboxvlating, EC 1.1.1.39) was assayed by the method of Ochoa (1955) to investigate whether it interfered with the measurement of malate oxidation by EC 1.1.1.37. The assay medium contained glvcine buffer, 0.1 M, p1-I 7.0; MnC&, 0.1 mM; NAD, 10 mM; homogenate, 0.2 mg of protein; and r.-malic acid, 10 mM. Measurements were made at 25 C and 340 nm. Lactate dehydrogenase (EC 1.1.1.27) was measured by the method of Shonk and
Boxer ( 1964). Th e assay medium contained triethanolamine/EDTA buffer, 0.05 M; NADH, 0.234 mM; homogenate, 0.4 mg of protein; and pyruvic acid, 15 mM. Measurements were made at 25 C and 340 nm. Units of activity have been expressed as micromoles of NAD or NADH reduced or oxidized per milligram of protein of homogenate per minute. Kinetic properties of malate dehydrogenase (EC 1.1.1.37) in terms of pH optima, substrate optima, cofactor, optima, and effects of temperature have also been investigated. All experiments were repeated using the same homogenate at least three times. Electrophoresis. Polyacrylamide-gel electrophoresis was conducted by the method of Toombs and Akroyd (1967) using 7.5% gels and 0.05 ml of homogenate (4 mg/mI of protein). A current of 2.5 mA/gel was applied for l-2 hr and the gels were stained by the method of Zee and Zinkham (1968). Control gels containing homogenate minus substrate were also employed. Electron microscopy. The purity and subcellular integrity of the mitochondrial fraction was confirmed using an AEl Corinth 275 microscope using the method described by Probert and Lwin (1974). RESULTS
Kinetic Properties of Malate Dehydrogenase ( EC 1.I .I .37) Malate oxidation was optimal at pH 10 in the supernatant and mitochondrial fractions while oxalacetate reduction was optimal in both fractions at pH 9.0 using both glycine and triethanolamine/EDTA buffers. Maximum activity of oxalacetate reduction was seen at 0.3 mM substrate and 0.235 mM NADH in both fractions. For malate oxidation, maximal activity was recorded at 7.4 mM malate and 1 mM NAD (Table I). Increasing the substrate concentration above 0.5 mM oxalacetate and 15 mM malate caused inhibition of activity in both fractions (Figs. 1 and 2). The Michaelis constants for both substrate and coenzyme are given in Table I.
Fasciola hepatica:
MALATE ‘I’ABLIS
Some
Catalytic
Properties
of Malate
I
and Lactate
Dehydrogenase
Mitochondrial ()I,Lim:il )‘I1
hlalate (1X
dehydrogenase 1.1.1.37)
0.u - malate Xlalate + 0A.Z S.\I)H N.\I)
0
in b’usciola
fraction
h~pa/ica
Supematant
fraction
-A-,,, (.I!)
!J.O 3.6 x 10.0 1.67 X 9.0 1.17 x 10.0 0.3 X 0‘1,~ ,-.--___-_ .\I:lliltc
i.ll
91
DEHYDROGENASE
1 x
mx&lxLl specific wtivity 10-j 10-s 10-j 10-a
mnlate -o.\.\
8.02 0.72 3.02 O.i2
* 0.27 f 0.12 f0.27 f0.12
I’lI
KS-Y.0 10.0 8.SY.O 10.0
h-In (-If)
5.2 2 1.17 0.45
X x X x
hlaximal specific activity 10-5 10-a 10-E 10-s
OhA - mslate .\\la!att? + ()A.\
= 1.2
II)-?
0.03;
i.0
0
0
SJ.0
3.26 0.495 8.26 0.495
3~0.26 f 0.08 f 0.26 * 0.08
= 6.6
1 x
10 ”
n.oi.5
7.5 x
10 3
0.050
Linearity of activity was seen with increasing homogenate concentration (up to 2 mg of protein) for both fractions. Maximal activity was 3.26 f 0.26 units/mg of protein/min and 3.02 + 0.27 for the supernatant and mitochondrial fractions, respectively, during oxalacetate reduction (n = S), and 0.495 + 0.08 and 0.72 f 0.12 for the supernatant and mitochondrial fractions, respectively, during malate oxidation (n = 5). The ratio of oxalacetate reduction to malate oxidation for the supernatant was 6.6: 1 and for the mitochondrial fraction 4.211. During oxalacetate reduction, activity in-
creased linearly with temperature up to 50 C with inactivation at 60 C. However, 20% of the original activity remained at 60 C in the supernatant (Fig. 3) while complete inactivation occurred in the mitochondrial fraction. During malate oxidation complete inactivation occurred in both fractions at 50 C (Fig. 3). When the two fractions were subjected to 50 C for 30 min prior to assay both reactions showed a loss of at least 60% of the optimal activity in the supernatant while 90-98% was destroyed in the mitochondrial fraction (Fig. 4).
FIG. 1. The effect of substrate concentration on malate dehydrogenase ( oxalacetate reduction) activity in supematant ( O-O ) and mitochondrial (O-O) fractions of Fmciolu hepaticu.
FIG. 2. The effect of substrate concentration on malate dehydrogenase (malate oxidation) activity in supematant ( O-O ) and mitochondrial (O0) fractions of Fusciolu heputica.
92
PROBERT
Electrophoretic Dehydrogenase
Properties
0
0/
A
PO/ O-5-
./*
o$“/ . t,j,,
c*0 .?
20
LWIN
of Malate
The whole homogenate displayed three bands of activity and these three bands also appeared in the supernatant fraction while only the first band appeared in the mitochondrial fraction. The thermostability of these bands was measured by exposure to 50 C for 15, 30, 45, and 60 min prior to electrophoresis. The first band disappeared from both fractions after a 30-min exposure at 50 C while bands 2 and 3 disappeared from the supernatant fraction after 60 min. Malate dehydrogenase (decarboxylating EC 1.1.1.39). The activity of this enzyme was low (Table I) and its optimal pH was lower than that for malate dehydrogenase (EC 1.1.1.37). It is unlikely therefore that the activity of this enzyme would interfere with the measurement of EC 1.1.1.37. Lactate dehydrogenase ( EC 1 .I J.27). It is claimed that lactate dehydrogenase activ-
1.5 -
AND
FIG. 4. The effect of treatment at 50 C for varying periods of time, prior to incubation, on malate dehydrogenase activity of supematant and mitochondrial fractions of Fusciola hepatica. Malate oxidation ( w); oxalacetate reduction (a).
ity may interfere with the assay of malate dehydrogenase since oxalacetate is liable to undergo decarboxylation to pyruvate (Wilcock and Goldberg 1972). However, since the activity of this enzyme was low (Table I ) and the K, to pyruvate high, it is unlikely that it would interfere with the measurement of oxalacetate reduction. To offset any possibility of decarboxylation of substrate freshly prepared oxalacetate solution was always used. Electron Microscopy
30
40
50
60
The mitochondrial fraction was seen to contain large numbers of mitochondria with no evidence of contamination with other organelles. Furthermore, the mitochondria were intact with no disrupted membranes. DISCUSSION
0’
I
n 20
30 Temperature
40
50
60
‘C
FIG. 3. The effect of temperature on malate dehydrogenase activity in supernatant ( 0-0 ) and mitochondrial (0-O) fractions of F~scioh~ hepaticn. (A) Malate oxidation; (B) oxalacetate reduction.
The kinetic and electrophoretic properties of malate dehydrogenase in F. hepatica indicate that there are two types of malate dehydrogenase present, one in the mitochondria and the other within the cytoplasm. Although the pH optima were similar, inhibition by high concentrations of malate was more marked in the case of the supernatant enzyme. The reverse was seen
Fusciola
hepatica:
MALATE
with high concentrations of oxalacetate where the mitochondrial enzyme was most affected. The supernatant enzyme was less susceptible to heat inactivation than that of the mitochondria. Elcctrophoresis of both fractions adds fur&r support to this view since three bands were seen in the supernatant and only one in the mitochondrial fraction. The heat-labile band in the supernatant corrcsponds with the mitochondrial band. Since the mitochondrial membranes were seen to be intact under electron microscopy it is unlikely that this band has resultcad from enzyme leaking into the supcrnatant during the fractionation procedure. Multiple electrophoretic forms of mitochondrial and supernatant malate dehydrogenase have been reported in many mammalian tissues (Thorne et a,!. 1963; Henderson 1964; Kitto and Kaplan 1966; Patton et al. 1967). Two isoenzymes of malate dehydrogenase were reported in whole homogenates of Schistosoma manso,zi adults and ccrcariae by Conde-de1 Pinto et al. (1966) while Oya et al. (1970) showed four isoenzymcs. Zce and Zinkham (1968) demonstrated four isoenzymcs of malate dehydrogenase in Ascaris suum, one in the mitochondria and three in the supernatant. The presence of three isoenzymes in the supernatant and one in the mitochondria of F. hepatica shows surprising similarity with the situation in Ascaris suum. Prichard and Schofield (1968b) and De Zoeten et al. (1969) suggested that in F. hepatica following conversion of glucose to phosphoenol pyruvate by glycolysis, it is carboxylated to oxalacetate, which in turn is reduced to malate by malate dehydrogenase and eventually via fumarate to succinate with the subsequent yield of ATP. A similar hypothesis was proposed by Bueding and Saz (1968) and Saz and Lescure (1969) for Ascaris lumbricoides. The higher activity of oxalacetate reduction compared with malate oxidation in F. hepatic supports this view. However, the high activity of malate oxidation in the mitochondria
9:;
DEHYDROCENASE
suggests the operation of a TCA cycle in F. hepatica. Prichard and Schofield (1968a) and De Zoeten et al. (1969) showed that all the necessary enzymes for such a cycle wcrc present in F. hepatica while Bryant and Williams (1962) and Thorsell (1963) showed all the intermediates. It seems likely therefore that both pathways for energy production occur in this parasite. It remains to be seen which of these pathways is the more important. Prichard and Schofield (1968a) suggested that the TCA cycle is of minor importance in F. hepatica. The importance of malate oxidation and oxalacctatc reduction in the metabolism of F. hepatica has already been shown by Lwin and Probcrt (1976) who demonstrated the inhibition of this enzyme by hexachlorophene and oxyclozanide at concentrations which were sufficient to kill flukes in &To. ACKNOWLEDGMENT T. Lwin would like to acknowledge the financial support of the Burmese Government to enable him to study in Britain. REFERENCES C., AND WILLIAMS, J. P. G. 1962. Some aspects of the metabolism of the liver fluke Faxiola hepatica L. Experimental Parasitology 12,372-376. BUEDING, E., AND SAZ, H. J. 1968. Pyruvatekinase and phosphoenolpyruvate carboxykinase activities of Ascaris muscle, Hymenolepis diminuta and Schistosoma mansoni. Comparative BioBRYANT,
chemistry and Physiology 24, 511-518. CONDE-DEL PINTO, E., PEREZ-VILAR, M., CITRONRIVERA, A. A., AND SENARIS, R. 1966. Studies on S. mnnsoni. I. Malic and lactic dehydrogenase of adult worms and cercariae. Experimental Parasitology 18, 320-326. DE ZOETEN, L. W., POSTHUMA, D., AND TIPKER,
J. 1969. Intermediary metabolism fluke F. hepatica. I. Biosynthesis acid. Hoppe-Seyler’s Zeitschrift
of the liver of propionic
fiir Phy.yiologische Chemie 350, 683-690. HENDERSON, N. S. 1964. Isoenzymes of malate dehydrogenase. Federation Proceedings 23, 487. KABAT, E. A., AND MAYER, M. M. 1961. “Experimental Immunochemistry.” Springfield, Ill.
Charles C Thomas,
94 Krrro, tion drial
PROBERT
C. B., AND KAPLAN, N. 0. 1966. Purificaand properties of chicken heart mitochonand supernatant malic dehydrogenase.
Biochemistry
5, 3966-3980.
Lwm, T., AND PROBERT, A. J. 1975. Effect of certain fasciolicides on malate dehydrogenase activity of Fusciokz hepatica: A possible biochemical mode of action of hexachlorophene and oxyclozanide. Pesticide Science 6, 121-128. OCHOA, S. 1955. Malic dehydrogenase from pig heart. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, pp. 739-792. Academic Press, New York. OYA, H., HAYASHI, H., AND Aoxr, T. 1970. Comparison of malate dehydrogenase isoenzymes between adult worms of S. mansoni and S. iaponicum. In “Recent Advances in Research on Filariasis and Schistosomiasis in Japan,” pp. 393403. University of Tokyo Press. PATTON, G. W., METS, L., AND VILLEE, C. A. 1967. Malic dehydrogenase isoenzymes. Distribution in developing nucleate and anucleate halves of sea urchin eggs. Science 156, 400401. PENNOIT-DE COOMAN, E., AND VAN GREMBERGEN, G. 1942. “Vergelijkend Onderzock van het Fermenten-system bij vrijlevende en parasitaire Plathelminthen,” Vol. 4, pp. 7-77. Verslagen en voorstellan van der K. Vlaamse Academic voor Wetenschappen, leheven en schone Kunsten van Belgie, Brussels. PRICHARD, R. K., AND SCHOFIELD, P. J. 1968a. A comparative study of the TCA cycle enzymes in F. hepaticu and rat liver. Comparative Biochemistry and Physiology 25, 1005-1019. PRICHARD, R. K., AND SCOFIELD, P. J. 1968b. The
AND
LWIN
metabolism fluke F.
of phosphoenol
pyruvate
heputicu. Biochimicu 170, 63-76.
in the adult
et Biophysicu Actu
PROBERT, A. J., AND Lwm, T. 1974. Kinetic properties, and location of non-specific phosphomonoesterases in subcellular fractions of FuscioZu
heputicu. Experimental
Parasitology 35, 253-261.
H. J., AND LESCURE, C. L. 1969. of phosphoenolpyruvate carboxykinase enzyme in anaerobic fermentation by A. lumbricoides. Compurutive
SAZ,
The
function and malic of succinate
Biochemistry
and Physiology 30, 49-60. SHONK, C. E., AND BOXER, G. E. 1964. patterns in human tissue. I. Methods determination of glycolytic enzymes.
Enzyme for the
Cancer
Research 24, 709-721. TIIORNE, C. J. R., CROSSMAN, L. I., AND KAPLAN, N. C. 1963. Starch gel electrophoresis of malate dehydrogenase. Biochimica et Biophysics Actu 73, 193-203. THORSELL, W. 1693. Biochemical studies on the liver fluke F. heputicu L. Actu Chemicu Scundinuvicu 17, 884. TOOMBS, M. P., AND AKROYD, P. 1967. Acrylamide gel electrophoresis. Shundon Instrument Applicu-
tions 18. WILCOCK, A. R., AND GOLDBERG, D. M. 1972. Kinetic determinations of malate dehydrogenase eliminating problems due to spontaneous conversion of oxalacetate to pyruvate. Biochemical Medicine 6, 116-126. ZEE, D. S., AND ZINKHAM, W. H. 1968. Malate dehydrogenase in A. suum. Characterization; ontogeny and genetic control. Archives of Biochemistry and Biophysics. 126, 574-584.
41,89-94
PARASITOLOGY
fascia/a hepatica: Electrophoretic
( 1977)
The Subcellular Distribution and Kinetic Properties of Malate Dehydrogenase A. J.
Department
of Applied
(Accepted
PROBERT
AND
T. LWIN 1
Zoology, Bangor,
University Gwynedd,
College U.K.
for
publication
2 March
of North
and
Wales,
1976)
PROBEI~T, A. J., AND LWIS, T. 1977. Fasciola Izepatica: The subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase. Experimental Parasitology 41, 89-94. The optimal pH for oxalacetate reduction was 9.0 for the mitochondrial and supernatant enzyme. The optimal pH for malate oxidation was 10 for both fractions. Maximal activity during oxalacetate reduction was 3.26 * 0.26 pmoles of NADH oxidized/ mg of protein/min and 3.02 * 0.27 for the supernatant and mitochondrial fractions, respectively. Maximal malate oxidation was 0.495 f 0.08 and 0.72 f 0.12 (not statistically different) for the supernatant and mitochondrial fractions, respectively. High concentrations of malate inhibited activity to a greater extent in the supernatant fraction while oxalacetate inhibited activity to a greater extent in the mitochondrial fraction. The supematant malate dehydrogenase was more heat stable than the mitochrondrial enzyme. Electrophoresis showed three isoenzymes of malate dehydrogenase in whole homogenates of Fascioka hepatica and all three were represented in the supernatant while only one band was seen in the mitochondrial fraction. The presence of two types of malate dehydrogenase, one in the supematant with three isoenzymes and one in the mitochondria with one isoenzyme, is proposed. INDEX
DESCRIPTORS:
and EC 1.1.1.39); geneity; Subcellular
FascioZu hepatica; Trematode; Malate dehydrogenase Lactate dehydrogenase (EC 1.1.1.27); Biochemistry; distribution; Electrophoretic properties.
INTRODUCTION
Pennoit-De Cooman and van Grembergen (1942) first reported the presence of malate dehydrogenase in Fasciola hepatica and this was confirmed later by Prichard and Schofield (1968a). It has been postulated by Prichard and Schofield (1968b) and De Zoeten et al. (1969) that malate dehydrogenase catalyzes the reduction of oxalacetate to a greater extent than malate oxidation in F. hepatica. This leads to the production
of
1 Present Parasitology, Veterinary
Copyright All rights
succinnte,
which
is coupled
address: Department of Veterinary Institute of Animal Husbandry and Science, Rangoon, Burma.
6 1977 by Academic of reproduction in any
Press, Inc. form reserved.
(EC Enzyme
1.1.1.37 hetero-
with ATP synthesis, thereby generating energy by reversal of some steps in the tricarboxylic acid cycle, rather than by oxidative processes. However, evidence for the presence of all the enzymes and intcrmediates necessary for a functional TCA cycle in F. hepatica has been reported (Thorsell 1963; Prichard and Schofield 1968a; De Zoeten et al. 1969). In view of the important position which malate dehydrogenase occupies in the metabolism of F. hepatica, studies have been undertaken on the subcellular distribution and the kinetic and electrophoretic properties of this enzyme.
ISSN
0014-4894
90
PROBERT AND LWIN MATFJUALSANDMETHODS
Preparation of homogenates. Ten percent (w/v) homogenates of adult flukes from cattle were prepared according to the method of Probert and Lwin (1974). Mitochondrial and supematant fractions were prepared by the method of Prichard and Schofield ( 1968a). The protein concentration was measured in each preparation by the biuret method (Kabat and Mayer 1961). Preparations were diluted with 0.25 M sucrose to given the desired concentrations. Enzyme assays. Assays were conducted spectrophotometrically using a Cecil ultraviolet spectrophotometer using thermoregulated cuvettes and on external pen recorder. All assayswere carried out at 25 C in a final volume of 3 ml and, except where otherwise stated, the reaction mixture was preincubated at the experimental temperature for 5 min prior to the addition of the substrate. Malate dehydrogenase (EC 1.1.1.37) was measured by the method of Shonk and Boxer ( 1964). The assaymedium contained triethanolamine plus EDTA buffer, 0.05 M; NADH, 0.234 mM; homogenate, 0.05 mg of protein; and cis-oxalacetic acid (neutralized), 0.33 mM. The oxidation of malate was assayed by the method of Prichard and Schofield ( 1968a). The medium contained glycine-NaOH buffer, 0.1 M; NAD, 1 mM; homogenate, 0.1 mg of protein; and L-malic acid (neutralized), 10 mM. The reaction in each case was started by addition of substrate and activity recorded from the increase or decrease in extinction at 340 nm. Malate dehydrogenase ( decarboxvlating, EC 1.1.1.39) was assayed by the method of Ochoa (1955) to investigate whether it interfered with the measurement of malate oxidation by EC 1.1.1.37. The assay medium contained glvcine buffer, 0.1 M, p1-I 7.0; MnC&, 0.1 mM; NAD, 10 mM; homogenate, 0.2 mg of protein; and r.-malic acid, 10 mM. Measurements were made at 25 C and 340 nm. Lactate dehydrogenase (EC 1.1.1.27) was measured by the method of Shonk and
Boxer ( 1964). Th e assay medium contained triethanolamine/EDTA buffer, 0.05 M; NADH, 0.234 mM; homogenate, 0.4 mg of protein; and pyruvic acid, 15 mM. Measurements were made at 25 C and 340 nm. Units of activity have been expressed as micromoles of NAD or NADH reduced or oxidized per milligram of protein of homogenate per minute. Kinetic properties of malate dehydrogenase (EC 1.1.1.37) in terms of pH optima, substrate optima, cofactor, optima, and effects of temperature have also been investigated. All experiments were repeated using the same homogenate at least three times. Electrophoresis. Polyacrylamide-gel electrophoresis was conducted by the method of Toombs and Akroyd (1967) using 7.5% gels and 0.05 ml of homogenate (4 mg/mI of protein). A current of 2.5 mA/gel was applied for l-2 hr and the gels were stained by the method of Zee and Zinkham (1968). Control gels containing homogenate minus substrate were also employed. Electron microscopy. The purity and subcellular integrity of the mitochondrial fraction was confirmed using an AEl Corinth 275 microscope using the method described by Probert and Lwin (1974). RESULTS
Kinetic Properties of Malate Dehydrogenase ( EC 1.I .I .37) Malate oxidation was optimal at pH 10 in the supernatant and mitochondrial fractions while oxalacetate reduction was optimal in both fractions at pH 9.0 using both glycine and triethanolamine/EDTA buffers. Maximum activity of oxalacetate reduction was seen at 0.3 mM substrate and 0.235 mM NADH in both fractions. For malate oxidation, maximal activity was recorded at 7.4 mM malate and 1 mM NAD (Table I). Increasing the substrate concentration above 0.5 mM oxalacetate and 15 mM malate caused inhibition of activity in both fractions (Figs. 1 and 2). The Michaelis constants for both substrate and coenzyme are given in Table I.
Fasciola hepatica:
MALATE ‘I’ABLIS
Some
Catalytic
Properties
of Malate
I
and Lactate
Dehydrogenase
Mitochondrial ()I,Lim:il )‘I1
hlalate (1X
dehydrogenase 1.1.1.37)
0.u - malate Xlalate + 0A.Z S.\I)H N.\I)
0
in b’usciola
fraction
h~pa/ica
Supematant
fraction
-A-,,, (.I!)
!J.O 3.6 x 10.0 1.67 X 9.0 1.17 x 10.0 0.3 X 0‘1,~ ,-.--___-_ .\I:lliltc
i.ll
91
DEHYDROGENASE
1 x
mx&lxLl specific wtivity 10-j 10-s 10-j 10-a
mnlate -o.\.\
8.02 0.72 3.02 O.i2
* 0.27 f 0.12 f0.27 f0.12
I’lI
KS-Y.0 10.0 8.SY.O 10.0
h-In (-If)
5.2 2 1.17 0.45
X x X x
hlaximal specific activity 10-5 10-a 10-E 10-s
OhA - mslate .\\la!att? + ()A.\
= 1.2
II)-?
0.03;
i.0
0
0
SJ.0
3.26 0.495 8.26 0.495
3~0.26 f 0.08 f 0.26 * 0.08
= 6.6
1 x
10 ”
n.oi.5
7.5 x
10 3
0.050
Linearity of activity was seen with increasing homogenate concentration (up to 2 mg of protein) for both fractions. Maximal activity was 3.26 f 0.26 units/mg of protein/min and 3.02 + 0.27 for the supernatant and mitochondrial fractions, respectively, during oxalacetate reduction (n = S), and 0.495 + 0.08 and 0.72 f 0.12 for the supernatant and mitochondrial fractions, respectively, during malate oxidation (n = 5). The ratio of oxalacetate reduction to malate oxidation for the supernatant was 6.6: 1 and for the mitochondrial fraction 4.211. During oxalacetate reduction, activity in-
creased linearly with temperature up to 50 C with inactivation at 60 C. However, 20% of the original activity remained at 60 C in the supernatant (Fig. 3) while complete inactivation occurred in the mitochondrial fraction. During malate oxidation complete inactivation occurred in both fractions at 50 C (Fig. 3). When the two fractions were subjected to 50 C for 30 min prior to assay both reactions showed a loss of at least 60% of the optimal activity in the supernatant while 90-98% was destroyed in the mitochondrial fraction (Fig. 4).
FIG. 1. The effect of substrate concentration on malate dehydrogenase ( oxalacetate reduction) activity in supematant ( O-O ) and mitochondrial (O-O) fractions of Fmciolu hepaticu.
FIG. 2. The effect of substrate concentration on malate dehydrogenase (malate oxidation) activity in supematant ( O-O ) and mitochondrial (O0) fractions of Fusciolu heputica.
92
PROBERT
Electrophoretic Dehydrogenase
Properties
0
0/
A
PO/ O-5-
./*
o$“/ . t,j,,
c*0 .?
20
LWIN
of Malate
The whole homogenate displayed three bands of activity and these three bands also appeared in the supernatant fraction while only the first band appeared in the mitochondrial fraction. The thermostability of these bands was measured by exposure to 50 C for 15, 30, 45, and 60 min prior to electrophoresis. The first band disappeared from both fractions after a 30-min exposure at 50 C while bands 2 and 3 disappeared from the supernatant fraction after 60 min. Malate dehydrogenase (decarboxylating EC 1.1.1.39). The activity of this enzyme was low (Table I) and its optimal pH was lower than that for malate dehydrogenase (EC 1.1.1.37). It is unlikely therefore that the activity of this enzyme would interfere with the measurement of EC 1.1.1.37. Lactate dehydrogenase ( EC 1 .I J.27). It is claimed that lactate dehydrogenase activ-
1.5 -
AND
FIG. 4. The effect of treatment at 50 C for varying periods of time, prior to incubation, on malate dehydrogenase activity of supematant and mitochondrial fractions of Fusciola hepatica. Malate oxidation ( w); oxalacetate reduction (a).
ity may interfere with the assay of malate dehydrogenase since oxalacetate is liable to undergo decarboxylation to pyruvate (Wilcock and Goldberg 1972). However, since the activity of this enzyme was low (Table I ) and the K, to pyruvate high, it is unlikely that it would interfere with the measurement of oxalacetate reduction. To offset any possibility of decarboxylation of substrate freshly prepared oxalacetate solution was always used. Electron Microscopy
30
40
50
60
The mitochondrial fraction was seen to contain large numbers of mitochondria with no evidence of contamination with other organelles. Furthermore, the mitochondria were intact with no disrupted membranes. DISCUSSION
0’
I
n 20
30 Temperature
40
50
60
‘C
FIG. 3. The effect of temperature on malate dehydrogenase activity in supernatant ( 0-0 ) and mitochondrial (0-O) fractions of F~scioh~ hepaticn. (A) Malate oxidation; (B) oxalacetate reduction.
The kinetic and electrophoretic properties of malate dehydrogenase in F. hepatica indicate that there are two types of malate dehydrogenase present, one in the mitochondria and the other within the cytoplasm. Although the pH optima were similar, inhibition by high concentrations of malate was more marked in the case of the supernatant enzyme. The reverse was seen
Fusciola
hepatica:
MALATE
with high concentrations of oxalacetate where the mitochondrial enzyme was most affected. The supernatant enzyme was less susceptible to heat inactivation than that of the mitochondria. Elcctrophoresis of both fractions adds fur&r support to this view since three bands were seen in the supernatant and only one in the mitochondrial fraction. The heat-labile band in the supernatant corrcsponds with the mitochondrial band. Since the mitochondrial membranes were seen to be intact under electron microscopy it is unlikely that this band has resultcad from enzyme leaking into the supcrnatant during the fractionation procedure. Multiple electrophoretic forms of mitochondrial and supernatant malate dehydrogenase have been reported in many mammalian tissues (Thorne et a,!. 1963; Henderson 1964; Kitto and Kaplan 1966; Patton et al. 1967). Two isoenzymes of malate dehydrogenase were reported in whole homogenates of Schistosoma manso,zi adults and ccrcariae by Conde-de1 Pinto et al. (1966) while Oya et al. (1970) showed four isoenzymcs. Zce and Zinkham (1968) demonstrated four isoenzymcs of malate dehydrogenase in Ascaris suum, one in the mitochondria and three in the supernatant. The presence of three isoenzymes in the supernatant and one in the mitochondria of F. hepatica shows surprising similarity with the situation in Ascaris suum. Prichard and Schofield (1968b) and De Zoeten et al. (1969) suggested that in F. hepatica following conversion of glucose to phosphoenol pyruvate by glycolysis, it is carboxylated to oxalacetate, which in turn is reduced to malate by malate dehydrogenase and eventually via fumarate to succinate with the subsequent yield of ATP. A similar hypothesis was proposed by Bueding and Saz (1968) and Saz and Lescure (1969) for Ascaris lumbricoides. The higher activity of oxalacetate reduction compared with malate oxidation in F. hepatic supports this view. However, the high activity of malate oxidation in the mitochondria
9:;
DEHYDROCENASE
suggests the operation of a TCA cycle in F. hepatica. Prichard and Schofield (1968a) and De Zoeten et al. (1969) showed that all the necessary enzymes for such a cycle wcrc present in F. hepatica while Bryant and Williams (1962) and Thorsell (1963) showed all the intermediates. It seems likely therefore that both pathways for energy production occur in this parasite. It remains to be seen which of these pathways is the more important. Prichard and Schofield (1968a) suggested that the TCA cycle is of minor importance in F. hepatica. The importance of malate oxidation and oxalacctatc reduction in the metabolism of F. hepatica has already been shown by Lwin and Probcrt (1976) who demonstrated the inhibition of this enzyme by hexachlorophene and oxyclozanide at concentrations which were sufficient to kill flukes in &To. ACKNOWLEDGMENT T. Lwin would like to acknowledge the financial support of the Burmese Government to enable him to study in Britain. REFERENCES C., AND WILLIAMS, J. P. G. 1962. Some aspects of the metabolism of the liver fluke Faxiola hepatica L. Experimental Parasitology 12,372-376. BUEDING, E., AND SAZ, H. J. 1968. Pyruvatekinase and phosphoenolpyruvate carboxykinase activities of Ascaris muscle, Hymenolepis diminuta and Schistosoma mansoni. Comparative BioBRYANT,
chemistry and Physiology 24, 511-518. CONDE-DEL PINTO, E., PEREZ-VILAR, M., CITRONRIVERA, A. A., AND SENARIS, R. 1966. Studies on S. mnnsoni. I. Malic and lactic dehydrogenase of adult worms and cercariae. Experimental Parasitology 18, 320-326. DE ZOETEN, L. W., POSTHUMA, D., AND TIPKER,
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C. B., AND KAPLAN, N. 0. 1966. Purificaand properties of chicken heart mitochonand supernatant malic dehydrogenase.
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Lwm, T., AND PROBERT, A. J. 1975. Effect of certain fasciolicides on malate dehydrogenase activity of Fusciokz hepatica: A possible biochemical mode of action of hexachlorophene and oxyclozanide. Pesticide Science 6, 121-128. OCHOA, S. 1955. Malic dehydrogenase from pig heart. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 1, pp. 739-792. Academic Press, New York. OYA, H., HAYASHI, H., AND Aoxr, T. 1970. Comparison of malate dehydrogenase isoenzymes between adult worms of S. mansoni and S. iaponicum. In “Recent Advances in Research on Filariasis and Schistosomiasis in Japan,” pp. 393403. University of Tokyo Press. PATTON, G. W., METS, L., AND VILLEE, C. A. 1967. Malic dehydrogenase isoenzymes. Distribution in developing nucleate and anucleate halves of sea urchin eggs. Science 156, 400401. PENNOIT-DE COOMAN, E., AND VAN GREMBERGEN, G. 1942. “Vergelijkend Onderzock van het Fermenten-system bij vrijlevende en parasitaire Plathelminthen,” Vol. 4, pp. 7-77. Verslagen en voorstellan van der K. Vlaamse Academic voor Wetenschappen, leheven en schone Kunsten van Belgie, Brussels. PRICHARD, R. K., AND SCHOFIELD, P. J. 1968a. A comparative study of the TCA cycle enzymes in F. hepaticu and rat liver. Comparative Biochemistry and Physiology 25, 1005-1019. PRICHARD, R. K., AND SCOFIELD, P. J. 1968b. The
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Enzyme for the
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