Thermal dependence in kinetic properties of lactate dehyrogenase from the African clawed toad, Xenopus laevis

Comp. Biochem. Physiol., Vol. 66B, pp. 459 to 466 Pergamon Press Ltd 1980, Printed in Great Britain

THERMAL D E P E N D E N C E IN KINETIC PROPERTIES OF LACTATE D E H Y D R O G E N A S E FROM THE AFRICAN CLAWED TOAD, X E N O P U S L A E V I S KATSUJI TSUGAWA Department of Biological Sciences, Osaka Women's University, Daisen-cho, Sakai 590 Japan

(Received 21 December 1979) Abstract--1. LDH of toad heart tissue exhibited lower apparent Km (pyruvate) and activation energy than those of liver and skeletal muscle. 2. In every tissue examined K,. values were decreased as reaction temperature reduced and "positive thermal modulation" was exhibited. 3. Cold acclimation did not affect the K,.-temperature relation of liver enzyme, although enzyme activity and K,, values were somewhat higher in cold-acclimated toads. 4. Ql0 of lactate oxidation activity of the two more anodic isozymes of liver LDH, which decreased relatively by cold acclimation, was higher than that of the least migrating isozyme, indicating a physiological meaning of isozyme pattern change during thermal acclimation.

MATERIALS AND METHODS

INTRODUCTION

Since the isozyme pattern of lactate dehydrogenase (LDH) was demonstrated to change with thermal acclimation of goldfish (Hochachka, 1965), the role of isozyme compositions and kinetic properties of enzymes has been emphasized in thermal acclimation of ectothermic animals (see review by Hochachka & Somero, 1973). In an aquatic amphibia, the South African clawed toad, Xenopus laevis, relative activity of anodic isozymes of L D H to cathodic one was found to be decreased by cold acclimation. The change was demonstrated in cells cultured in vitro at low temperature, too, suggesting the possibility of the "autonomous adaptation" of tissue cells to environmental temperature not mediated by the superposed regulatory system(s) of organisms (Tsugawa, 1976). The possible physiological meanings of such changes in isozyme patterns of toad L D H could be expected to be explained by different thermal dependences of kinetic properties of isozymes. Temperature coefficients of reactions catalyzed by five isozymes of rabbit L D H were directly related to the electrophoretic mobilities of isozymes (Plagemann et al., 1961). Liver L D H from cold- and warm-acclimated brook trout were reported to display different Kin-temperature relation, with low Km values occurring at their respective acclimation temperatures (Hochachka & Lewis, 1971) and the correlation between isozyme pattern and temperature dependence of K,, was suggested in isocitrate dehydrogenase of trout (Moon & Hochachka, 1972). In the present paper thermal dependence of apparent K,, of pyruvate for L D H and enzyme activity were examined in liver tissues of cold- and warmacclimated toads and, in order to clarify the possible physiological meanings of the changes in isozyme pattern with thermal acclimation, the differences in kinetic properties of L D H among isozymes were estimated from the comparison of tissue enzymes exhibiting different isozyme patterns. C,B.P. 66/4B--B

Young adult Xenopus laevis, hatched from eggs in the laboratory, were kept at 18-23°C under 13L:llD photoperiod. They were acclimated to warm (25 + I°C) or cold (14 + I°C) environment in examination for thermal acclimation. They were fed on liver of chick or cattle twice a week at 18-25°C or once at 14°C. Wistar rats were purchased from Jikken Dobutsu Kansai Kenkyu-sho, Osaka. The whole liver and heart and thigh muscle were chosen as enzyme sources, because of the characteristic isozyme patterns of their LDH. After an animal was killed by decapitation, one or two kinds of tissues were quickly excised and immediately washed in ice-cold phosphate-buffered saline (PBS: 8.0g NaC1, 0.2 g KCI, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 1 1. of water for mammal and the same solution diluted to 1.3131. for toad). Then they were blotted thoroughly and weighed. They were homogenized in 80-150 vol of ice-cold PBS using Potter-Elvehjem homogenizer with Teflon pestle and centrifuged at 13,000 y for 20min. When two tissues were examined, one was kept unhomogenized in ice-cold PBS until the kinetic assay of enzyme from the other tissue was completed. Supernatant fraction was immediately used for kinetic study. For measurement of enzyme activities alone (Table 1), toad livers were homogenized in 0.1 M Tris-HC1 buffer, pH 7.0, containing 2 mM ethylenediamineteraacetate (EDTA) and 2mM glutathione reduced (GSH) and centrifuged at 20,000 # for 20 min. LDH activity was assayed spectrophotometrically using Hitachi 124 double beam spectrophotometer. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.4, 0.1 mM NADH, varying concentrations of sodium pyruvate and 5 to 20/zl of enzyme solution. NADH and pyruvate solutions were prepared freshly each day to reduce the possibility of inhibitor formation. Reaction medium in a cuvette was stirred continuously and the temperature of medium was controlled at a constant temperature (+0.3°C) using a specially modified cuvette holder equipped with thermodules in iron block and a thermister sensor in the cuvette of I cm light path (Komatsu Electronics Co., Tokyo). Initial rates were estimated by measuring the rate of NADH oxidation at 340 nm from 30 to 90 sec after the initiation of the reaction by the addition of enzyme solution.

459

460

KATSUJI TSUGAWA Table 1. LDH activities in livers of toads acclimated to 14°C and 25°C for 1-2 months Acclimation

~mol NADH o x i d i z e d / m i n

temperature

per g t i s s u e

per mg p r o t e i n

250C

283 + 15

4.32 + 0.22

14°C

319 + 15

4.80 + 0.19

Activities were measured at 25°C with 2 mM pyruvate as substrate. Values are mean + SEM of 12 animals. Activities were proportional to the amounts of enzyme solution added over the range of activity of 0.005 to 0.07/xmols NADH oxidized per min with 0.1-0.2 mM pyruvate as substrate at 25°C. About 15 hr were required to measure the activities at various concentrations of substrate at different temperatures, but storage of enzyme solution within 22 hr at 0°C did not cause either of decrease in enzyme activity or significant change in apparent K,, value. Activity of LDH prepared in Tris-HCl buffer was slightly lower (83%) than the activity of enzyme prepared in PBS. But the comparison between activities in two acclimation groups was quite possible as the activity was proportional to the volume of enzyme solution added over the range of enzyme concentrations examined and was stable at least for 10 hr without any decrease of activity. Protein was determined by a modified Lowry's procedure, with bovine serum albumin (Sigma, Type III) as the standard. Polyacrylamide disc gel electrophoresis was performed as in the previous paper (Tsugawa, 1976). Supernatant fraction of homogenate at 900 # for 10 min was used as source of electrophoresis. Bands of LDH activity were visualized by incubating gels at different temperatures and relative activity of isozymes was calculated from the area of each peak demonstrated by densitometry.

(a)

Effect of acclimation on LDH activity in toad liver L D H activities, expressed as /xmols N A D H oxidized/min per g tissue wet weight or per mg protein of enzyme preparation, did not change significantly with cold acclimation of toad for 4-6 weeks, although some increase in activities could be suspected (Table 1).

Thermal dependence of the rate of pyruvate reduction Figure 1 shows the relationship between the activities of toad L D H and the concentrations of substrate at various temperatures. L D H activities were not saturated at high temperatures over the range of pyruvate concentrations examined, while at lower temperatures they were saturated at relatively low concentration of substrate. Heart enzyme was more susceptible to high concentration of substrate than the enzymes from the other tissues examined. A Lineweaver-Burk plot of L D H activity demonstrates more clearly the temperature-dependent substrate inhibition (Fig. 2). In every tissue examined substrate inhibition appeared at the concentration as

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Fig. 1. Effects of reaction temperatures and substrate concentrations on activities of LDH from toad heart (a), muscle (b) and liver (c) tissues. Velocity v is expressed in #mol NADH oxidized/min per mg protein. I1: 35°C, O: 25°C, O: 15°C, A: 5°C.

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Fig. 2. Lineweaver-Burk plots at different temperatures for toad LDH from heart (a), muscle (b) liver (c). O: 40°C, O: 35°C, A: 30°C, &: 25°C, V: 20°C, V: 15°C, r-3: 10°C, I : 5°C. low as 1 mM at 25°C and it increased with decreasing temperature. In rat examined as a comparison, heart enzyme was more susceptible to high concentration of pyruvate than liver enzyme. Substrate inhibition was increased as the temperature lowered. Arrhenius plots of log Vma x calculated from Lineweaver-Burk plot vs 1/T exhibited a straight line in every enzyme prepared. On the other hand, plot of actual activities measured, even at high concentration of substrate, showed bending of the line and as the concentration of substrate added lowered, the slope approached 0 (Figs 3 and 4). Activation energy (Ea) for heart enzyme was lower than the value of muscle or liver enzyme in toad (Table 2).

Table 2. Activation energy (kcal/mol) of LDH from various tissues of toad and rat LDH from

activation energy

X. laevis

liver 25°C-acclimated 14°C-acclimated

12.1 + 0.21 (9)* ]2.0 ~ 0.49 (4) 12.2 ~ 0.06 (5)

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12.2, 12.3 I0.7, I0.9

liver heart

l l . l , 12.4 12.8, 13.7

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Thermal dependence of apparent Km of pyruvate The apparent Km values calculated from the Lineweaver-Burk plot increased in every tissue from both toad and rat as the reaction temperature increased. Km value for rat heart LDH was lower and more temperature-dependent than that for liver enzyme (Fig. 5). In Xenopus the value for heart enzyme was about half of those for liver and muscle enzymes at every temperature examined (Fig. 6). Acclimation of toad to cold did not affect the Kin-temperature relation in liver enzyme. K , value for liver enzyme showed a tendency to increase with cold acclimation at every temperature examined except 2°C, but the difference between two acclimation groups was statistically significant only at 15°C (P < 0.05 by Mann-Whitney U-test; Fig. 6b).

Thermal dependence of lactate oxidation activities by different isozymes of toad LDH Figure 7 shows the zymograms of LDH stained at different temperatures after polyacrylamide disc gel electrophoresis. The activity of anodic isozymes decreased relatively as the incubation temperature dropped. The ratio of Qlo value of anodic isozyme(s) to that of the least anodic one can be calculated from relative activities of isozymes at different temperatures by the following equation: Qlo(A) _ A,+ lo/A, _ (A/C)t+ lo Q 1o(C) C, + 1o/C, (A/C), where A and C are activities of anodic and cathodic (the least anodic) isozymes, respectively, and A/C relative activity of anodic iso~yme(s) to cathodic one calculated from the areas of peaks demonstrated by densitometry. The suffix, t and t + 10, shows the temperature of incubation of gels. By this means, Qto value of heart-type isozyme was shown to be 1.5-fold higher than that of the least

462

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migrating isozyme over the temperature range of 15~5°C. The two more anodic isozymes of liver L D H were sensitive to reaction temperature 1.4-fold as compared with the least anodic one (Fig. 8).

findings are in agreement with those for purified enzymes or isozymes from the same (Wieland, 1959) and other mammals (Latner & Skillen, 1968) and indicate that the comparison of crude preparations

DISCUSSION

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In the present experiment with rat LDH, heart enzyme (predominantly, anodic isozyme) was shown to be more sensitive to high concentration of substrate and to have lower Km value of pyruvate than liver enzyme (predominantly, cathodic ones). These

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°C Fig. 6. Thermal dependences of apparent Km of pyruvate for toad LDH. (a) heart (open symbols) and muscle (closed ones) enzymes. A sort of symbols for each tissue represents the value of enzyme preparation from the same individual. (b) liver enzymes from toads acclimated to 25 + I°C (0) and 14 + I°C (O) for 2-6 months. Each value represents the mean (+SEM) of 4-6 toads except at 2°C where the values of 2 animals were substituted for SEM. with different isozyme compositions gives some, but significant informations upon kinetic properties of isozymes. LDH of X . laevis has isozyme constituent distinct from mammalian ones. Liver enzyme was composed of typical 5 isozymes, of which 2 cathodic isozymes were major ones in skeletal muscle. Heart tissue contained predominantly the "heart-type isozyme" which moved more rapidly towards anode than any isozyme of liver enzyme did (Claycomb & Villee, 1971 ; Lyra et al., 1976; Tsugawa, 1976). Kinetic studies with X. laevis LDH revealed high sensitivity to pyruvate inhibition and low Km value of pyruvate in heart enzyme as compared with liver and muscle enzymes (Figs 1, 2 and 6) as in many LDH from rat (Fig. 5; Wieland, 1959) and other mammals ( L a t n e r & Skiilen, 1968) and from poikilotherms (snake, De Burgos et al., 1973; brook trout and lake trout, Wuntch & Goldberg, 1970; Rana pipiens, Goldberg & Wuntch, 1967; Eby et al., 1973; Levy & Salth, 1974; Enig et al., 1976). The lower K,, value for heart-type isozyme seems to be also the case in this animal despite of unique isozyme pattern of toad LDH. E,, however, was lower in heart LDH, thus, hearttype isozyme, than liver and muscle enzymes in contrast with the cases of many other animals such as Rana temporaria (Olsson, 1975), snake (De Burgos et al., 1973), rat (present data) and other mammals (Plagemann et al., 1961; De Burgos et al., 1973; Olsson, 1975) with the exception of tuna (Hochachka & Somero, 1968) and cod (Olsson, 1975) ones.

463

LDH from every tissue examined exhibited a sharp decrease in K= of pyruvate with decreasing temperature to 5°C (Figs 5 and 6). Similar results had been reported in R. pipiens in which Km value for heart enzyme was decreased to one-third as the assay temperature declined from 25 to 5°C (Levy & Salth, 1971). On the contrary, Eby et al. (1973) and Enig et al. (1976) reported that Km for heart LDH, but not muscle LDH, was almost temperature-independent. As expected from the temperature-dependent changes of Km value and thermal independence of E~ over the range of biological temperature, Ql0 of LDH activity approached to unity as the concentration of substrate lowered. As pyruvate concentrations of 0.08 to 0.15 raM, which may be considered physiological (Black et al., 1961; Freed, 1971), the slopes of Arrhenius plots became flat around 30°C (Fig. 3). De Burgos et al. (1973) reported supraoptimal thermal compensation in snake LDH at low concentration of substrate (0.05 mM pyruvate). Qlo values of LDH activity of toad liver, muscle and heart tissues were 1.15, 1.07 and 1.25 at the same concentration in physiological temperature range (15-25°C), contrasting with the high Ql0 values of Vm,x (2.0, 2.1 and 1.9), respectively. At lower temperatures the values became somewhat higher as a result of higher sensitivity to pyruvate inhibition. Wuntch et al. (1970) suggested that substrate inhibition of rat LDH might not occur in vivo where the enzyme concentration is high. If at physiological enzyme concentration the pyruvate inhibition of toad LDH is much less than that observed with the enzyme solution used, Qlo value at low temperature would drop to or under the value of moderate temperature range and almost perfect thermal compensation would function effectively over a wide range of temperature in which toad might be experienced. These results indicate that in toad tissue "positive thermal modulation" (Hochachka & Somero, 1973) could play an important role in immediate thermal compensation of LDH activity. Such mechanisms, however, could have physiological significance only at acute temperature drop ol short duration when the cellular concentration of substrate does not change. If the concentration of substrate would be lowered in proportion to the decrease in Km value during prolonged cold exposure of tissue cells, then one could not expect the thermal compensation by means of Km change. Frankel et al. (1966' reported a decrease of blood pyruvate concentratio~ to about 70~o of 25°C-exposed turtle by exposure tc 5°C for 1 day. Freed (1971) found that the concentration of pyruvate in muscle of goldfish acclimated tc 5°C was only one-sixth of that in 25°C-acclimated fish. Therefore, it would be too hasty judgement tc conclude that the temperature-dependent change ir Km would have physiological significance to compen. sate the decrease in LDH activity caused by drop o: reaction temperature during prolonged cold acclima. tion of toad until the intracellular concentration o substrate available to enzyme is demonstrated t( change little. On the other hand, the increase of abou 10~o in LDH activity of liver tissue in cold-acclimate( toads (Table 1), though statistically not significant might be an indication of compensatory change t( reduce a decrease in enzyme activity due to drop o reaction temperature.

464

KATSUJI TSUGAWA

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37

45

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25

37

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Fig. 7. Isozyme patterns of toad liver LDH (a) and mixed preparation of heart and skeletal muscle enzymes (b). Polyacrylamide disc gels were incubated with reaction mediuni at 4 different temperatures indicated.

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4,5

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Fig. 8. Comparison of thermal dependences of lactate oxidation activities of anodic and cathodic isozymes of toad LDH, (a) mixed preparation of heart and muscle enzymes, (b) liver enzyme. The value A/C expresses relative activity of the anodic isozyme(s) (indicated as A at the bottom of the figure) to the cathodic (the least anodic) one (indicated as C). Parallel lines were given for two independent experiments (O and O).

Thermal dependence of X. laevis LDH activity Km values of pyruvate were reported to change (in:rease, decrease or shift of Kin-minimum temperature) Nith thermal acclimation in liver LDH of brook trout Hochachka & Lewis, 1971) and in muscle one of frog R. pipiens, Enig et al., 1976) and crayfish (Narita & ~oriuchi, 1979). On the other hand, in muscle LDH )f the long-jaw mudsucker (Somero, 1973) and heart ;nzyme of frog (R. pipiens, Enig et al., 1976) the value ~as not affected by cold acclimation. In toad, accli~aation to cold did not bring about a change in theraaal dependence of Km for liver enzyme (Fig. 6b). K,~ ¢alue for liver enzyme showed a tendency to increase with cold acclimation as in muscle LDH of goldfish Yamawaki & Tsukuda, 1979). The change was com3arable with the change due to a drop of reaction :emperature of about 2°C and apparently compen;ated 20% of the change in Km value which would aave been brought about by cold exposure of toads. It should he noted that no significant difference in dnetic properties could be detected between toad LDH from liver and skeletal muscle tissues. It may 9e, in part, attributed to the fact that the isozymes zommon with muscle enzyme assumed a high proporIion of enzyme activity in liver tissue (Claycomb & Villee, 1971; Lyra et al., 1976; Tsugawa, 1976). But Ihe difference in K,~ values between LDH from toad heart and muscle tissues, between which almost no zommon isozyme was contained, was less than the difference between those from rat tissues in which zonsiderable amounts of common isozymes were contained (Figs 5 and 6). Therefore, it seems probable that variation of kinetic properties among isozymes of toad LDH is less than the variation among those of mammalian LDH. The failure in detecting differences in both Km value of pyruvate and its thermal dependence between liver and muscle LDH (Fig. 6) makes it difficult to explain directly the change of isozyme pattern caused by cold acclimation from the expected isozyme-specific Kr, value or its thermal dependence, as the change in isozyme pattern in liver tissue by cold acclimation was less marked as compared with the difference between the two tissues (Tsugawa, 1976). Investigation of lactate oxidation activity of the different isozymes of toad LDH revealed high Qxo values in the anodic isozymes as compared with the cathodic one (Fig. 8). The value of lactate oxidatton by heart-type isozyme was about 1.5-fold of that of muscle-type isozyme (the least anodic one) and that the anodic components of liver enzyme also exhibited almost similar Q10 value to that of heart one. The higher Qlo values of lactate oxidation activity catalized by anodic isozymes were also the case in the enzymes of other species of Anura, Rana nioromaculata, R. catesbeiana and Hyla arborea japonica (Tsugawa, unpublished data) and in liver LDH of goldfish (Tsukuda, 1975). The difference in Qlo values among isozymes could clarify a possible physiological significance of the changes in isozyme pattern during physiological adaptation to temperature of toad cells in situ and in vitro (Tsugawa, 1976). It was the anodic isozymes of LDH, the more cold-sensitive ones, that decreased relatively by cold adaptation of cells. It could be of great advantage to maintenance of metabolic activity in cold environment to which animals were exposed.

465

Acknowledgements--The author wishes to thank Mrs E. Kitano and Miss K. Motooka for their assistance for determinating LDH activity in Table 1 and Dr H. Tsukuda for her helpful criticisms of the manuscript. REFERENCES

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tive activities of liver lactate dehydrogenase isozymes in goldfish acclimated to different temperatures. Comp. Biochem. Physiol. 52B, 343-345. WIELAND TH., PFLEIDERER G. & ORTANDERL F. (1959) Uber die Verschiedenheit der Milchs~iuredehydrogenasen III. Vergleiche der Milchs~iuredehydrogenasen aus verschiedenen Rattenorganen. Biochem. Z. 331, 103-109. WUNTCH T. & GOLDBERG E. (1970) A comparative physico-chemical characterization of lactate dehydrogenase: Isozymes in brook trout, lake trout and their hybrid splake trout. J. exp. Zool. 174, 233 252. Wt~NTCH T., CHEN R. F. & VESELL E. S. (1970) Lactate dehydrogenase isozymes: Kinetic properties at high enzyme concentrations. Science 167, 63-65. YAMAWAKI H. & TSUKUDA H. (1979) Significance of the variation in isozymes of liver lactate dehydrogenase with thermal acclimation in goldfish II. Effect of pH. Comp. Biochem. Physiol. 62B, 95 99.