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Adaptation of enzymes to temperature: Lactate and malate dehydrogenases from platypus and echidna
Comp. Biochem. Physiol., 1973, VoL 44B, pp. 363 to 370.PergamonPress. Printed in Great Britain
ADAPTATION OF ENZYMES TO TEMPERATURE: LACTATE A N D MALATE DEHYDROGENASES FROM PLATYPUS A N D ECHIDNA JOHN BALDWIN and M I C H A E L A L E K S I U K * t Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2600, Australia (Received 30 May 1972)
Abstract--1. The apparent K,n of muscle LDH for pyruvate and of liver supernatant MDH for oxaloacetate and NADH increases with increasing assay temperature over the range 10-40°C. 2. Arrhenius plots of log Vnmt vs. temperature -x for these enzymes are linear from 10 to 40°C. 3. At low substrate concentrations temperature has little effect upon reaction rates over the range 25-35°C, but reaction rates fall with decreasing temperature below 20°C. 4. It is proposed that these effects of temperature on reaction rate may be of adaptive significance in stabilizing reaction rates in active animals subjected to fluctuating body temperatures and in the slowing down of metabolism during hibernation. INTRODUCTION RECENT studies of the thermal properties of enzyme systems in poikilothermic animals have revealed clearly adaptive mechanisms by which catalytic and regulatory properties can be maintained in the face of markedly different and often rapidly fluctuating environmental temperatures (Hochachka & Somero, 1958; Somero & Hochachka, 1958; Behrisch & Hochachka, 1959; Somero, 1969; Baldwin & Hochachka, 1970; Hochachka & Lewis, 1970; Moon & Hochachka, 1971). As an extension of these studies we have examined the effects of temperature upon muscle lactate dehydrogenases (LDH; E.C. 1.1.1.27; L-lactate; NAD oxidoreductase) and liver supernatant malate dehydrogenases (MDH; E.C. 1.1.1.37; L-malate; NAD oxidoreductase) in the monotremes platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus). These animals are of particular interest from a thermoregulatory viewpoint, as they are subjected to fluctuating body temperatures and, at least in the case of echidna, undergo hibernation. Data on body temperatures of echidnas in the field (Miklovho-Maclay, 1883 ; Griffiths, 1968; Augee et al., 1970), in the laboratory (Schmidt-Nielsen et aL, 1966; Augee & Ealey, 1968) and in outdoor enclosures (Augee et aL, * Visiting Research Fellow. ~"Present address: Zoology Department, University of Manitoba, Winnipeg~ Manitoba, Canada. 363
364
JOHN BALDWINAND MICHAI~LALEKSIUK
1970) indicate that body temperatures of active animals generally lie within the range 23-32°C. During prolonged periods of low ambient temperature the animals can become torpid for periods of at least 9 days with body temperatures closely paralleling environmental temperatures down to 10°C and possibly lower. Little is known of the response of the aquatic platypus to fluctuating environmental temperatures. S m y t h (1970) demonstrated the ability of animals held in the laboratory to maintain stable body temperatures of about 31°C at air t e m p e r a tures from 0.5 to 25°C. However, animals were unable to maintain body t e m p e r a tures at this level when subjected to a water temperature of 11.5°C, with a drop in b o d y temperature of up to 10°C occurring after 4 hr. While it has been claimed that the platypus hibernates, definitive data are lacking (Barrett, 1944; Fleay, 1944). MATERIALS AND M E T H O D S
Experimental animals Animals (Ornithorhynchus anatinus, Tachyglossus aculeatus) were kindly provided by Dr. M. Griffith, C.S.I.R.O., Division of Wildlife Research, Gungahlin, and by Mr. Peter Temple-Smith, Department of Zoology, Australian National University. Skeletal muscle and heart were stored at -20°C. Liver tissue was removed immediately after death and processed at once without freezing.
Preparation of enzymes Skeletal muscle, heart muscle and liver were homogenized in ice-cold 50 mM sodium phosphate buffer, pH 7"4 (1 g tissue/4 ml), and centrifuged at 30,000 g for 30 min in a Sorvall RC-2B refrigerated centrifuge. The supernatants were used as a source of L D H and M D H without further purification.
Electrophoresis Enzyme extracts were examined by starch gel electrophoresis on 11"5% gels using a 200 mM phosphate-citrate tank buffer pH 7-0. Gels were prepared in a 1 : 20 dilution of this buffer and run for 18 hr at 4°C with a voltage gradient of 6 V/cm. L D H and M D H activity were detected by described methods (Markert & Ursprung, 1962) with sodium lactate and sodium malate as substrates.
Assay of enzyme activity Activity was assayed with a Gilford 2400 recording spectrophotometer coupled to a circulating water-bath. The reaction was followed by measuring the oxidation of N A D H at 340 m/z. The reaction mixture contained sodium pyruvate or sodium oxaloacetate, NADH and 50 mM sodium phosphate buffer pH 7-4, in a total volume of 3 ml. The reaction was started by the addition of 10/~I of suitably diluted enzyme preparation following thermal equilibrium of the reaction mixture.
RESULTS AND DISCUSSION
Electrophoretic forms of L D H Skeletal muscle and heart extracts of platypus and echidna give electrophoretic profiles (Fig. 1) which are more complex than those found in most of the higher
ADAPTATION OF ENZYMES TO TEMPERATURE
365
vertebrates. While both monotremes basically show the typical binomial distribution of the five LDH tetramers H4, H3M , H~M2, HM 3 and M 4 (LDH1-5 respectively), each tetramer, with the exception of H4, is accompanied by up to 2 sub-bands. In a study to be published elsewhere, it has been shown that the platypus possesses a third subunit type in addition to M and H, which provides an explanation of the sub-banding pattern. The echidna LDH system is at present under investigation and it appears probable that an additional subunit is also present. Effect of assay temperature on the apparent Km of pyruvate for skeletal muscle L D H The relationships between apparent K m for pyruvate and assay temperature are shown in Fig. 2. The increase in apparent K m with increasing temperature for LDH enzymes from both animals are similar to K m temperature effects that have been reported for a wide range of enzymes from poikilotherms (Hochachka & Somero, 1968; Somero, 1969; Baldwin & Hochachka, 1970; Moon & Hochachka, 1971).
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Temperature, °C FIG. 2. Effect of assay t e m p e r a t u r e o n t h e a p p a r e n t K m of p y r u v a t e for L D H e n z y m e s f r o m p l a t y p u s (©) a n d e c h i d n a (@) skeletal muscle. L D H was assayed at p y r u v a t e c o n c e n t r a t i o n s in the r a n g e 0"05-5 m M at a c o n s t a n t s a t u r a t i n g N A D H c o n c e n t r a t i o n of 0.1 r a M . K m values were d e t e r m i n e d f r o m d o u b l e - r e c i p r o c a l plots (v - I vs. pyruvate-1).
The adaptive significance of this phenomenon is thought to lie in the ability of thermally induced changes in enzyme-substrate affinity (as measured by the reciprocal of Kin) to counteract the effect of fluctuating thermal kinetic energy upon reaction rates at low and probable physiological substrate concentration (Hochachka & Somero, 1968). This rate stabilization effect at low substrate concentrations (Fig. 3) (K m levels of pyruvate and NADH) occurs with both the platypus and echidna muscle LDH enzymes over the temperature range 25-35°C, but at lower temperatures reaction rates fall with decreasing temperature.
366
J O H N B A L D W I N AND M I C H A E L A L E I 6 I U K
These effects of assay temperature upon reaction velocity are summarized in Table 1. TABLE
1 - - E F F E C T OF ASSAY TEMPERATURE UPON REACTION RATES AT SATURATING AND AT M I N I M U M K m LEVELS OF SUBSTRATE FOR L D H AND M D H ENZYMES FROM PLATYPUS AND ECHIDNA
Temperature range
(°c) Platypus LDH
0~o v , ~ 0~o K~ (S)
10-20 25-35 10-20 25-35 10-20 25-35 10-20 25-35
Echidna LDH Platypus MDH Echidna MDH
3.2 2.7 3.2 2.7 1.7 1"6 2.2 1"9
1.5 0.9 1-4 1"0 1"6 1-1 1.6 1.0
18
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,'o
FIG. 3. Effect of assay temperature on the activity of LDH enzymes from platypus (©) and echidna (0) skeletal muscle at low substrate concentrations. Standard assay conditions with 0"1 mM pyruvate and 0"01 mM NADH.
Effect of assay temperature on the maximum velocity of skeletal muscle L D H Arrhenius plots of log V,,ax vs. T -z for the muscle L D H enzymes are shown in Fig. 4. In each ease log Vm~x increases linearly with temperature from 10 to 40°C, yielding energies of activation of 19 and 17 kcal respectively for the platypus and echidna L D H enzymes. T h e linear nature of the Arrhenius plots supports the hypothesis that rate stabilization at low substrate concentrations is achieved through alterations in enzyme-substrate affinity. I f the observed temperature independence of reaction rate from 25 to 35°C resulted from a thermally induced change in the rate limiting step for the overall reaction, which in turn might influence reaction velocities at low substrate concentration, the Arrhenius plots would be non-linear,
Pm
Ph
Em
Eh
i '
FIG. 1. Electrophoretic forms of platypus and echidna skeletal muscle and heart LDH. Pm, platypus muscle; Ph, platypus heart; Era, echidna muscle; Eh, echidna heart.
-
-I-
FIG. 5. Electrophoretic forms of platypus and echidna liver supernatant MDH. P, platypus; E, echidna.
ADAPTATION
OF ENZYMES
TO TEMPERATURE
367
unless two or more mutually compensatory changes occurred. Q10 values over various temperature ranges are given in Table 1, and show dearly that stabilization of reaction rate does not occur at saturating substrate concentrations. 1
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~20 ,
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330
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340
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350
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105/T, oK FIO. 4. Arrhenius plots of skeletal muscle LDH activity (log Vmax vs. T -t °K) for platypus (©) and echidna (Q).
Electrophoretic forms of liver supernatant M D H The electrophoretic forms of M D H obtained for platypus and echidna liver preparations are shown in Fig. 5. If it is assumed that monotreme M D H enzymes are structurally similar to those of other vertebrates, then the three bands obtained for platypus can be interpreted in terms of two subunit types giving rise to three dimeric isozymes. For echidna, only one band of M D H activity is present. While this could mean that echidna possesses only one subunit type, leading to a single dimerie enzyme, this observation alone is not sufficient to preclude the presence of additional subunit types producing isozymes which were not resolved under the experimental conditions employed. Effect of assay temperature on the apparent K m of oxaloacetate and N.4DH for liver supernatant M D H The relationships between apparent Km for oxaloacetate and NADH, and assay temperature, for the platypus and echidna M D H enzymes are shown in Figs. 6 and 7. For oxaloacetate the effect of assay temperature on apparent K m is essentially the same as that observed for pyruvate with the muscle L D H enzymes. The apparent K m increases with increasing temperature to give approximately a fourfold change
JOHNBALDWIN AND
368
MICHAEL ALEKSIUK
over the temperature range 10-40°C for both the platypus and echidna M D H enzymes. With NADH, however, the apparent K m is much less affected by temperature, increasing about 1.8 fold over the same temperature range in each case. 10
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FIG. 6. Effect of assay temperature on the apparent K,, of oxaloacetate for liver supernatant M D H enzymes from platypus (O) and echidna (0). M D H was assayed at oxaloacetate concentrations in the range 0"002-0"5 m M at a constant saturating N A D H concentration of 0"1 raM. K,~ values were determined from double-reciprocal plots (v -1 vs. oxaloacetate-1). ~c~ 3
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Temperature, °C FIc. 7. Effect of assay temperature on the apparent Km of N A D H for liver supernatant M D H enzymes for platypus (©) and echidna (O). M D H was assayed at N A D H concentrations in the range 0"001-0"325 m M at a constant saturating oxaloacetate concentration of 0"5 m M. Km values were determined from double-reciprocal plots (v -1 vs. NADH-1).
The effect of these changes in apparent K m upon reaction rates at low substrate concentrations are plotted in Fig. 8 and summarized in Table 1. As with the muscle L D H enzymes, reaction rates are held essentially constant from 25 to 35°C but fall significantly at temperatures below 20°C.
369
ADAPTATION OF ENZYMES TO TEMPERATURE
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Temperature, °C Fzo. 8. Effect of assay temperature on the activity of liver supernatant MDH enzymes from platypus (O) and echidna (0) at low substrate concentrations. Standard assay conditions with 0"02 mM oxaloacetate and 0"02 mM NADH.
Effect of assay temperature on maximum velocity of liver supernatant M D H Arrhenius plots of log Vmax vs. T -1 for platypus and echidna M D H enzymes (Fig. 9) are linear and essentially parallel over the temperature range 10-40°C, with an energy of activation of 16.5 kcal. Again it is apparent from Table 1 that the adaptive significance of thermally induced changes of apparent K m in stabilizing reaction rates is seen only at low substrate concentration approaching the probable physiological levels. 4
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FIO. 9. Arrhenius plots of liver supernatant MDH activity (log Vmax vs. T -1 °K) for platypus (O) and echidna (0). In summary, the data in Table 1 indicate that, over the range of body temperatures observed in active animals (25-35°C), reaction rates for L D H and M D H enzymes at K , , levels of substrates may be held essentially constant in the face of
370
JOHN BALDWINAND MICHAELALEKSIUK
fluctuating environmental temperatures, a situation analogous to the stabilizing effect of constant body temperature on enzyme systems in an efficient homeotherm. As temperatures fall below 20°C, reaction rates decrease, and it is tempting to speculate that this phenomenon may be of adaptive advantage in slowing down metabolism, thereby aiding the conservation of energy stores during hibernation.
Acknowledgements--We would like to thank Christine Hayes and Mary Aleksiuk for their excellent technical assistance. Financial support for travel of the junior author came from the Faculty of Graduate Studies and Research, University of Manitoba. REFERENCES
AuG~ M. L. & EALEYE. H. M. (1968) Torpor in the echidna Tachyglossus aculeatus. J. Mammal. 49, 446-454. AuG~ M. L., EALEYE. H. M. & SPENCERH. (1970) Biotelemetric studies of temperature regulation and torpor in the echidna Tachyglossus aculeatus. J. Mammal. 51, 561-570. BALDWINJ. & HOCHnC~ P. W. (1970) Functional significance of isoenzymes in thermal acclimatisation: acetylcholinesterase from trout brain. Biochem. J. 116, 883-887. B~T C. (1944) The Platypus. Roberton & Mullens, Melbourne. BEHRISCHH. W. & HOCHACHKAP. W. (1969) Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow trout fructose diphosphatase. Biochem. ~. 111, 287-295. FLEA',' D. (1944) Observations on the breeding of the platypus in captivity. Vict. Nat. 61, 8-14. GRI~ITHS M. (1968) Echidnas. Pergamon Press, London. HOCHACHg~P. W. & LEWISJ. K. (1970) Enzyme variants in thermal acclimation : trout liver citrate synthases, aT. biol. Chem. 245, 6567-6573. HOCHACHKAP. W. & SOMERO G. N. (1968) The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 27, 659-668. MARg~RTC. L. & UaSPRUNGH. (1962) The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Devl. Biol. 5, 363-381. MIKLOUHO-MACLAYN. (1883) Temperature of the body of Echidna hystrix. Proc. Linn. Soc. N.S.W. 8, 425. MOON T. W. & HOCHACHg~P. W. (1971) Temperature and enzyme activity in poikilotherms: isocitrate dehydrogenases in rainbow trout liver. Biochem. J. 123, 695-705. SCHMIDT-NIELSENK., DAWSONT. J. & CRAWFORDE. C., JR. (1966) Temperature regulation in the echidna. ~. cell Physiol. 67, 63-72. SMYTH D. M. (1970) Thermoregulation in the platypus Ornithorhynchus anatinus. B.Sc. Honours thesis, Zoology Department, The Australian National University, Canberra. SOMERO G. N. (1969) Enzymic mechanisms of temperature compensation: immediate and evolutionary effects of temperature on enzymes of aquatic poikilotherms. Am. Nat. 103, 517-530. SOMERO G. S. & HOCHACHKAP. W. (1968) The effect of temperature on catalytic and regulatory functions of pyruvate kinases of the rainbow trout and the Antarctic fish Trematomus bernacchii. Biochem. 3¢. 110, 395-399.
Key Word Index--LDH; MDH; enzymes-temperature; Ornithorhynchus anatinus; Tachyglossus aculeatus.
ADAPTATION OF ENZYMES TO TEMPERATURE: LACTATE A N D MALATE DEHYDROGENASES FROM PLATYPUS A N D ECHIDNA JOHN BALDWIN and M I C H A E L A L E K S I U K * t Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2600, Australia (Received 30 May 1972)
Abstract--1. The apparent K,n of muscle LDH for pyruvate and of liver supernatant MDH for oxaloacetate and NADH increases with increasing assay temperature over the range 10-40°C. 2. Arrhenius plots of log Vnmt vs. temperature -x for these enzymes are linear from 10 to 40°C. 3. At low substrate concentrations temperature has little effect upon reaction rates over the range 25-35°C, but reaction rates fall with decreasing temperature below 20°C. 4. It is proposed that these effects of temperature on reaction rate may be of adaptive significance in stabilizing reaction rates in active animals subjected to fluctuating body temperatures and in the slowing down of metabolism during hibernation. INTRODUCTION RECENT studies of the thermal properties of enzyme systems in poikilothermic animals have revealed clearly adaptive mechanisms by which catalytic and regulatory properties can be maintained in the face of markedly different and often rapidly fluctuating environmental temperatures (Hochachka & Somero, 1958; Somero & Hochachka, 1958; Behrisch & Hochachka, 1959; Somero, 1969; Baldwin & Hochachka, 1970; Hochachka & Lewis, 1970; Moon & Hochachka, 1971). As an extension of these studies we have examined the effects of temperature upon muscle lactate dehydrogenases (LDH; E.C. 1.1.1.27; L-lactate; NAD oxidoreductase) and liver supernatant malate dehydrogenases (MDH; E.C. 1.1.1.37; L-malate; NAD oxidoreductase) in the monotremes platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus). These animals are of particular interest from a thermoregulatory viewpoint, as they are subjected to fluctuating body temperatures and, at least in the case of echidna, undergo hibernation. Data on body temperatures of echidnas in the field (Miklovho-Maclay, 1883 ; Griffiths, 1968; Augee et al., 1970), in the laboratory (Schmidt-Nielsen et aL, 1966; Augee & Ealey, 1968) and in outdoor enclosures (Augee et aL, * Visiting Research Fellow. ~"Present address: Zoology Department, University of Manitoba, Winnipeg~ Manitoba, Canada. 363
364
JOHN BALDWINAND MICHAI~LALEKSIUK
1970) indicate that body temperatures of active animals generally lie within the range 23-32°C. During prolonged periods of low ambient temperature the animals can become torpid for periods of at least 9 days with body temperatures closely paralleling environmental temperatures down to 10°C and possibly lower. Little is known of the response of the aquatic platypus to fluctuating environmental temperatures. S m y t h (1970) demonstrated the ability of animals held in the laboratory to maintain stable body temperatures of about 31°C at air t e m p e r a tures from 0.5 to 25°C. However, animals were unable to maintain body t e m p e r a tures at this level when subjected to a water temperature of 11.5°C, with a drop in b o d y temperature of up to 10°C occurring after 4 hr. While it has been claimed that the platypus hibernates, definitive data are lacking (Barrett, 1944; Fleay, 1944). MATERIALS AND M E T H O D S
Experimental animals Animals (Ornithorhynchus anatinus, Tachyglossus aculeatus) were kindly provided by Dr. M. Griffith, C.S.I.R.O., Division of Wildlife Research, Gungahlin, and by Mr. Peter Temple-Smith, Department of Zoology, Australian National University. Skeletal muscle and heart were stored at -20°C. Liver tissue was removed immediately after death and processed at once without freezing.
Preparation of enzymes Skeletal muscle, heart muscle and liver were homogenized in ice-cold 50 mM sodium phosphate buffer, pH 7"4 (1 g tissue/4 ml), and centrifuged at 30,000 g for 30 min in a Sorvall RC-2B refrigerated centrifuge. The supernatants were used as a source of L D H and M D H without further purification.
Electrophoresis Enzyme extracts were examined by starch gel electrophoresis on 11"5% gels using a 200 mM phosphate-citrate tank buffer pH 7-0. Gels were prepared in a 1 : 20 dilution of this buffer and run for 18 hr at 4°C with a voltage gradient of 6 V/cm. L D H and M D H activity were detected by described methods (Markert & Ursprung, 1962) with sodium lactate and sodium malate as substrates.
Assay of enzyme activity Activity was assayed with a Gilford 2400 recording spectrophotometer coupled to a circulating water-bath. The reaction was followed by measuring the oxidation of N A D H at 340 m/z. The reaction mixture contained sodium pyruvate or sodium oxaloacetate, NADH and 50 mM sodium phosphate buffer pH 7-4, in a total volume of 3 ml. The reaction was started by the addition of 10/~I of suitably diluted enzyme preparation following thermal equilibrium of the reaction mixture.
RESULTS AND DISCUSSION
Electrophoretic forms of L D H Skeletal muscle and heart extracts of platypus and echidna give electrophoretic profiles (Fig. 1) which are more complex than those found in most of the higher
ADAPTATION OF ENZYMES TO TEMPERATURE
365
vertebrates. While both monotremes basically show the typical binomial distribution of the five LDH tetramers H4, H3M , H~M2, HM 3 and M 4 (LDH1-5 respectively), each tetramer, with the exception of H4, is accompanied by up to 2 sub-bands. In a study to be published elsewhere, it has been shown that the platypus possesses a third subunit type in addition to M and H, which provides an explanation of the sub-banding pattern. The echidna LDH system is at present under investigation and it appears probable that an additional subunit is also present. Effect of assay temperature on the apparent Km of pyruvate for skeletal muscle L D H The relationships between apparent K m for pyruvate and assay temperature are shown in Fig. 2. The increase in apparent K m with increasing temperature for LDH enzymes from both animals are similar to K m temperature effects that have been reported for a wide range of enzymes from poikilotherms (Hochachka & Somero, 1968; Somero, 1969; Baldwin & Hochachka, 1970; Moon & Hochachka, 1971).
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3
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0
0
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Temperature, °C FIG. 2. Effect of assay t e m p e r a t u r e o n t h e a p p a r e n t K m of p y r u v a t e for L D H e n z y m e s f r o m p l a t y p u s (©) a n d e c h i d n a (@) skeletal muscle. L D H was assayed at p y r u v a t e c o n c e n t r a t i o n s in the r a n g e 0"05-5 m M at a c o n s t a n t s a t u r a t i n g N A D H c o n c e n t r a t i o n of 0.1 r a M . K m values were d e t e r m i n e d f r o m d o u b l e - r e c i p r o c a l plots (v - I vs. pyruvate-1).
The adaptive significance of this phenomenon is thought to lie in the ability of thermally induced changes in enzyme-substrate affinity (as measured by the reciprocal of Kin) to counteract the effect of fluctuating thermal kinetic energy upon reaction rates at low and probable physiological substrate concentration (Hochachka & Somero, 1968). This rate stabilization effect at low substrate concentrations (Fig. 3) (K m levels of pyruvate and NADH) occurs with both the platypus and echidna muscle LDH enzymes over the temperature range 25-35°C, but at lower temperatures reaction rates fall with decreasing temperature.
366
J O H N B A L D W I N AND M I C H A E L A L E I 6 I U K
These effects of assay temperature upon reaction velocity are summarized in Table 1. TABLE
1 - - E F F E C T OF ASSAY TEMPERATURE UPON REACTION RATES AT SATURATING AND AT M I N I M U M K m LEVELS OF SUBSTRATE FOR L D H AND M D H ENZYMES FROM PLATYPUS AND ECHIDNA
Temperature range
(°c) Platypus LDH
0~o v , ~ 0~o K~ (S)
10-20 25-35 10-20 25-35 10-20 25-35 10-20 25-35
Echidna LDH Platypus MDH Echidna MDH
3.2 2.7 3.2 2.7 1.7 1"6 2.2 1"9
1.5 0.9 1-4 1"0 1"6 1-1 1.6 1.0
18
0~0
16
/ °
14 " ; 12
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8
~
6
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4
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2 0
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0
5
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10
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3'o
Temperature.°C
,'o
FIG. 3. Effect of assay temperature on the activity of LDH enzymes from platypus (©) and echidna (0) skeletal muscle at low substrate concentrations. Standard assay conditions with 0"1 mM pyruvate and 0"01 mM NADH.
Effect of assay temperature on the maximum velocity of skeletal muscle L D H Arrhenius plots of log V,,ax vs. T -z for the muscle L D H enzymes are shown in Fig. 4. In each ease log Vm~x increases linearly with temperature from 10 to 40°C, yielding energies of activation of 19 and 17 kcal respectively for the platypus and echidna L D H enzymes. T h e linear nature of the Arrhenius plots supports the hypothesis that rate stabilization at low substrate concentrations is achieved through alterations in enzyme-substrate affinity. I f the observed temperature independence of reaction rate from 25 to 35°C resulted from a thermally induced change in the rate limiting step for the overall reaction, which in turn might influence reaction velocities at low substrate concentration, the Arrhenius plots would be non-linear,
Pm
Ph
Em
Eh
i '
FIG. 1. Electrophoretic forms of platypus and echidna skeletal muscle and heart LDH. Pm, platypus muscle; Ph, platypus heart; Era, echidna muscle; Eh, echidna heart.
-
-I-
FIG. 5. Electrophoretic forms of platypus and echidna liver supernatant MDH. P, platypus; E, echidna.
ADAPTATION
OF ENZYMES
TO TEMPERATURE
367
unless two or more mutually compensatory changes occurred. Q10 values over various temperature ranges are given in Table 1, and show dearly that stabilization of reaction rate does not occur at saturating substrate concentrations. 1
"\.\ 5 4 I 3 2F
~20 ,
I
1
i
I
i
I
I
325
330
335
340
345
350
355
105/T, oK FIO. 4. Arrhenius plots of skeletal muscle LDH activity (log Vmax vs. T -t °K) for platypus (©) and echidna (Q).
Electrophoretic forms of liver supernatant M D H The electrophoretic forms of M D H obtained for platypus and echidna liver preparations are shown in Fig. 5. If it is assumed that monotreme M D H enzymes are structurally similar to those of other vertebrates, then the three bands obtained for platypus can be interpreted in terms of two subunit types giving rise to three dimeric isozymes. For echidna, only one band of M D H activity is present. While this could mean that echidna possesses only one subunit type, leading to a single dimerie enzyme, this observation alone is not sufficient to preclude the presence of additional subunit types producing isozymes which were not resolved under the experimental conditions employed. Effect of assay temperature on the apparent K m of oxaloacetate and N.4DH for liver supernatant M D H The relationships between apparent Km for oxaloacetate and NADH, and assay temperature, for the platypus and echidna M D H enzymes are shown in Figs. 6 and 7. For oxaloacetate the effect of assay temperature on apparent K m is essentially the same as that observed for pyruvate with the muscle L D H enzymes. The apparent K m increases with increasing temperature to give approximately a fourfold change
JOHNBALDWIN AND
368
MICHAEL ALEKSIUK
over the temperature range 10-40°C for both the platypus and echidna M D H enzymes. With NADH, however, the apparent K m is much less affected by temperature, increasing about 1.8 fold over the same temperature range in each case. 10
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Temperature, °C FIc. 7. Effect of assay temperature on the apparent Km of N A D H for liver supernatant M D H enzymes for platypus (©) and echidna (O). M D H was assayed at N A D H concentrations in the range 0"001-0"325 m M at a constant saturating oxaloacetate concentration of 0"5 m M. Km values were determined from double-reciprocal plots (v -1 vs. NADH-1).
The effect of these changes in apparent K m upon reaction rates at low substrate concentrations are plotted in Fig. 8 and summarized in Table 1. As with the muscle L D H enzymes, reaction rates are held essentially constant from 25 to 35°C but fall significantly at temperatures below 20°C.
369
ADAPTATION OF ENZYMES TO TEMPERATURE
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Effect of assay temperature on maximum velocity of liver supernatant M D H Arrhenius plots of log Vmax vs. T -1 for platypus and echidna M D H enzymes (Fig. 9) are linear and essentially parallel over the temperature range 10-40°C, with an energy of activation of 16.5 kcal. Again it is apparent from Table 1 that the adaptive significance of thermally induced changes of apparent K m in stabilizing reaction rates is seen only at low substrate concentration approaching the probable physiological levels. 4
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370
JOHN BALDWINAND MICHAELALEKSIUK
fluctuating environmental temperatures, a situation analogous to the stabilizing effect of constant body temperature on enzyme systems in an efficient homeotherm. As temperatures fall below 20°C, reaction rates decrease, and it is tempting to speculate that this phenomenon may be of adaptive advantage in slowing down metabolism, thereby aiding the conservation of energy stores during hibernation.
Acknowledgements--We would like to thank Christine Hayes and Mary Aleksiuk for their excellent technical assistance. Financial support for travel of the junior author came from the Faculty of Graduate Studies and Research, University of Manitoba. REFERENCES
AuG~ M. L. & EALEYE. H. M. (1968) Torpor in the echidna Tachyglossus aculeatus. J. Mammal. 49, 446-454. AuG~ M. L., EALEYE. H. M. & SPENCERH. (1970) Biotelemetric studies of temperature regulation and torpor in the echidna Tachyglossus aculeatus. J. Mammal. 51, 561-570. BALDWINJ. & HOCHnC~ P. W. (1970) Functional significance of isoenzymes in thermal acclimatisation: acetylcholinesterase from trout brain. Biochem. J. 116, 883-887. B~T C. (1944) The Platypus. Roberton & Mullens, Melbourne. BEHRISCHH. W. & HOCHACHKAP. W. (1969) Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow trout fructose diphosphatase. Biochem. ~. 111, 287-295. FLEA',' D. (1944) Observations on the breeding of the platypus in captivity. Vict. Nat. 61, 8-14. GRI~ITHS M. (1968) Echidnas. Pergamon Press, London. HOCHACHg~P. W. & LEWISJ. K. (1970) Enzyme variants in thermal acclimation : trout liver citrate synthases, aT. biol. Chem. 245, 6567-6573. HOCHACHKAP. W. & SOMERO G. N. (1968) The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 27, 659-668. MARg~RTC. L. & UaSPRUNGH. (1962) The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Devl. Biol. 5, 363-381. MIKLOUHO-MACLAYN. (1883) Temperature of the body of Echidna hystrix. Proc. Linn. Soc. N.S.W. 8, 425. MOON T. W. & HOCHACHg~P. W. (1971) Temperature and enzyme activity in poikilotherms: isocitrate dehydrogenases in rainbow trout liver. Biochem. J. 123, 695-705. SCHMIDT-NIELSENK., DAWSONT. J. & CRAWFORDE. C., JR. (1966) Temperature regulation in the echidna. ~. cell Physiol. 67, 63-72. SMYTH D. M. (1970) Thermoregulation in the platypus Ornithorhynchus anatinus. B.Sc. Honours thesis, Zoology Department, The Australian National University, Canberra. SOMERO G. N. (1969) Enzymic mechanisms of temperature compensation: immediate and evolutionary effects of temperature on enzymes of aquatic poikilotherms. Am. Nat. 103, 517-530. SOMERO G. S. & HOCHACHKAP. W. (1968) The effect of temperature on catalytic and regulatory functions of pyruvate kinases of the rainbow trout and the Antarctic fish Trematomus bernacchii. Biochem. 3¢. 110, 395-399.
Key Word Index--LDH; MDH; enzymes-temperature; Ornithorhynchus anatinus; Tachyglossus aculeatus.