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The effect of temperature on the kinetic properties of lactate dehydrogenase from embryos of various fish species
Comp. Biochem. Physiol. Vol. 107B, No. 4, pp. 593-595, 1994 Elsevier Science Ltd Printed in Great Britain 0305-0491/94 $6.00 + 0.00
Pergamon
The effect of temperature on the kinetic properties of lactate dehydrogenase from embryos of various fish species Olga S. Klyachko and Nikolai D. Ozernyuk N. K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., Moscow 117 808, Russia Temperature dependence of apparent K, for pyruvate of LDH from the embryos of seven species of ftshes was analyzed. For the embryos of rainbow trout, autumn cisco and least (Siberian) cisco, the K,. minima were observed at 5--8°C; for zebrafish, carp and goldfish they were at 25--28°C, and for the loach, at 13--16°C. During embryonic and larval development of rainbow trout proceeding at gradually increasing temperatures, the K, minimum was found to vary in parallel with the optimal temperature. For the embryos of rainbow trout whose development proceeds at constant temperature, the position of the K. minimum at the early gastrula stage and at hatching was the same. Key words: Lactate dehydrogenase; Salmo gairdneri; Coregonus autumnalis migratorius; Coregonus
sardinella; Misgurnus fossilis; Brachidanio rerio; Cyprinus carpio; Carassius auratus. Comp. Biochem. Physiol. 107B, 593-595, 1994.
Introduction Temperature adaptations of fish are related to the metabolic states of the organisms at optimal and unfavorable temperature conditions. Habitat temperature was shown to affect the apparent Michaelis constant (Kin) values for some enzymes, lactate dehydrogenase in particular, from various species of fish preferring different temperatures (Yancey and Somero, 1978; Hochachka and Somero, 1984; Coppes and Somero, 1990). Within the range of optimal temperatures the Ks values are usually minimal. These effects, however, missed the attention of investigators in the studies of ontogenesis that proceeds via various stages at different environmental temperatures. Therefore, we have examined the effect of habitat temperature on the Km value for pyruvate of LDH from embryos and larvae of various species of fish.
Correspondence to: N. D. Ozernyuk, N. K. Koltsov, Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., Moscow 117 808, Russia. Received 19 July 1993; accepted 22 September 1993.
M a t e r i a l s and M e t h o d s
Experimental animals Experiments were carried out on embryos of rainbow trout Salmo gairdneri, autumn cisco Coregonus autumnalis migratorius, least (Siberian) cisco Coregonus sardinella, loach Misgurnus fossilis, zebrafish Brachidanio rerio, carp Cyprinus carpio and goldfish Carassius auratus. Loach and goldfish eggs were obtained by chorionic gonadotropin injection of females (Neyfakh, 1959). Rainbow trout, autumn cisco, least (Siberian) eiseo, carp and zebrafish eggs were taken from naturally matured females.
Sample preparation and assay method Embryos at the early gastrula stage were homogenized in 0.1 M Tris--HCl buffer, pH 6.8, at 0-4°C and centrifuged at 15,000g for 10rain at 2°C. The supernatant was used for determination of enzyme activity (Cahn, 1964). Seven concentrations of pyruvate spanning the range 0.1-1.0 mM were used. Apparent K~ values were calculated by the method of Lineweaver-Burk
593
594
Olga S. Klyachko and Nikolai D. Ozernyuk
using double-reciprocal plots of velocity vs substrate concentration.
Results
K m (raM)
A 0.3
Km for eggs of various fish species The effect of assay temperature on the Km value for pyruvate of LDH from embryos of fish preferring different environmental temperatures was examined. For embryos at the early gastrula stage of various species the minimum of Km as a function of assay temperature differs distinctly (Fig. 1). For rainbow trout, autumn cisco and least (Siberian) cisco eggs, the minimum Km values were 5-8°C. For zebra fish, carp and goldfish embryos, the Km values show a minimum at 25-28°C, whereas for loach they were observed at 13-16°C. For the loach eggs the effect of temperature on Km is not quite as substantial.
0.2
0.1
0
K m (raM)
04[
0.3
IAc
/ 10
I 15
I 20
Temperature (°C)
Km for embryos and larvae of rainbow trout The effect of assay temperatures on the Km values for pyruvate from the rainbow trout embryos at different stages of development, and larvae also, was studied. The embryogenesis and larval development of rainbow trout in nature proceeds at different temperatures; the early stages are at relatively low temperature, the later ones at a higher temperature. For the embryos at the early gastrula stage the Km values showed a minimum at 5-7°C, whereas for those at hatching they were 10°C (Fig. 2.). For the
I 5
Fig. 2. Temperature effect on the K m for pyruvate of LDH from extracts of rainbow trout embryos at (A) early gastrula stage, (B) hatching, and (C) skeletal muscles of larvae.
skeletal muscles of larvae the Km minimum was at 10-13°C. The question arising is whether the disclosed kinetic features of LDH from embryos and larvae of rainbow trout are related to variation of the environmental temperature. The answer was found in the experiment on the development of embryos at constant ambient temperature (5°C). It was found that in this case the position of the Km minimum was the same for the embryos at the early gastrula stage as it was for those at hatching (Fig. 3). Hence, the variation of the Km minimum, illustrated in Fig. 2, appears to be related to variation of environmental temperature.
Discussion 0.2
0.1
0
;
1'0
,'5
;5
Temperature (*(2) Fig. I. Effect of temperature on apparent K m for pyruvate of LDH from extracts of embryos at the early gastrula stage. A: rainbow trout; B: least (Siberian) cisco; C: autumn cisco; D: loach; E: goldfish; F: carp; G: zebrafish.
For various fish species the minimum Km values are correlated with the optimal temperatures in nature (Hochachka and Somero, 1973; Yancey and Somero, 1978; Coppes and Somero 1990). This conclusion is supported for the embryos of fish preferring different environmental temperatures. For the embryos of rainbow trout, autumn cisco and least (Siberian) cisco, adapted to cold habitats, the apparent Kmvalues for pyruvate of LDH were at 5-8°C, whereas for the eggs of zebrafish, carp and goldfish, preferring warm habitats, this value was at 25-28°C. For the embryos of loach, adapted to moderate temperatures, the Km minima were observed at 13-17°C (Fig. 1). These results are consistent with the data for adult fishes
LDH in fish embryos
et al., 1975; Arai and Yamazaki, 1979, 1980).
K m (raM)
0.3
0.2
595
However, in the our experiment on the development of embryos at constant temperature it was found that the position of the Km minimum was the same for the embryos at both early and later stages of embryogenesis. Thus, the variation in the Km minimum appears to be related to the variation in environmental temperature.
I
References
0.1
o
1'0
1'5
Temperature (°C) Fig. 3. Effect of temperature on the Km for pyruvate of LDH from extracts of rainbow trout embryos developing at constant temperature (5°). A: early gastrula stage; B: hatching.
preferring different environmental temperatures (Yancey and Somero, 1978; Graves and Somero, 1982; Coppes and Somero, 1990). It is known that development of rainbow trout embryos in nature proceeds at increasing temperatures. Experiments with embryos incubated at constant temperature suggest that the increase in the Km minimum position during the development is related to the increase of habitat temperature. The change in the Km minimum position may by due to changes in enzyme isoforms during embryogenesis. Indeed, the changes in LDH isozyme patterns in embryogenesis of salmonid fish were reported (Wright
Arai K. and Yamazaki F. (1979) Developmental changes of lactate dehydrogenase (LDH) isozymes in the hybrid between masu salmon (Oncorhynchus masou) and pink salmon (O. gorbuscha). Bull. Fac. Fish. Hokkaido Univ. 30, 124-128. Arai K. and Yamazaki F. (1980) Developmental genetics on the lactate dehydrogenase (LDH) isozyme during early embryogenesis of salmonids. Bull. Fac. Fish. Hokkaido Univ. 31, 223-228. Cahn R. D. (1964) Developmental changes in embryonic enzyme patterns: the effect of oxidative substrates on lactic dehydrogenase in beating chick embryonic heart cell cultures. Devl BioL 9, 327-346. Coppes Z. L. and Somero G. N. (1990) Temperatureadaptive differences between the M4-1actate dehydrogenases of stenothermal and eurythermal sciaenid fishes. J. exp. Zool. 2.54, 127-131. Graves J. E. and Somero G. N. (1982) Electrophoretic and functional enzymic evolution in four species of Eastern Pacific barracudas from different thermal environments. Evolution 36, 97-106. Hochachka P. W. and Somero G. N. (1973) Strategies of Biochemical Adaptation. W. B. Saunders, Philadelphia. Hochachka P. W. and Somero G. N. (1984) Biochemical Adaption. Princeton University Press, Princeton, NJ. Neyfakh A. A. (1959) X-ray inactivation of nuclei as a method for studying their function in the early development of fishes. J. Embryol. exp. Morph. 2, 173-192. Wright J. E., Heckman R. and Aterton L. M. (1975) Genetics and developmental analysis of LDH isozymes in trout. In Isozymes-- 3. Developmental Biology (Edited by Markert C. L.), pp. 375-401. Academic Press, New York. Yancey P. H. and Somero G. N. (1978) Temperature dependence of intracellular pH: its role in the conservation of pyruvate apparent K,, values of vertebrate lactate dehydrogenase. J. comp. Physiol. 12,5, 129-134.
Pergamon
The effect of temperature on the kinetic properties of lactate dehydrogenase from embryos of various fish species Olga S. Klyachko and Nikolai D. Ozernyuk N. K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., Moscow 117 808, Russia Temperature dependence of apparent K, for pyruvate of LDH from the embryos of seven species of ftshes was analyzed. For the embryos of rainbow trout, autumn cisco and least (Siberian) cisco, the K,. minima were observed at 5--8°C; for zebrafish, carp and goldfish they were at 25--28°C, and for the loach, at 13--16°C. During embryonic and larval development of rainbow trout proceeding at gradually increasing temperatures, the K, minimum was found to vary in parallel with the optimal temperature. For the embryos of rainbow trout whose development proceeds at constant temperature, the position of the K. minimum at the early gastrula stage and at hatching was the same. Key words: Lactate dehydrogenase; Salmo gairdneri; Coregonus autumnalis migratorius; Coregonus
sardinella; Misgurnus fossilis; Brachidanio rerio; Cyprinus carpio; Carassius auratus. Comp. Biochem. Physiol. 107B, 593-595, 1994.
Introduction Temperature adaptations of fish are related to the metabolic states of the organisms at optimal and unfavorable temperature conditions. Habitat temperature was shown to affect the apparent Michaelis constant (Kin) values for some enzymes, lactate dehydrogenase in particular, from various species of fish preferring different temperatures (Yancey and Somero, 1978; Hochachka and Somero, 1984; Coppes and Somero, 1990). Within the range of optimal temperatures the Ks values are usually minimal. These effects, however, missed the attention of investigators in the studies of ontogenesis that proceeds via various stages at different environmental temperatures. Therefore, we have examined the effect of habitat temperature on the Km value for pyruvate of LDH from embryos and larvae of various species of fish.
Correspondence to: N. D. Ozernyuk, N. K. Koltsov, Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., Moscow 117 808, Russia. Received 19 July 1993; accepted 22 September 1993.
M a t e r i a l s and M e t h o d s
Experimental animals Experiments were carried out on embryos of rainbow trout Salmo gairdneri, autumn cisco Coregonus autumnalis migratorius, least (Siberian) cisco Coregonus sardinella, loach Misgurnus fossilis, zebrafish Brachidanio rerio, carp Cyprinus carpio and goldfish Carassius auratus. Loach and goldfish eggs were obtained by chorionic gonadotropin injection of females (Neyfakh, 1959). Rainbow trout, autumn cisco, least (Siberian) eiseo, carp and zebrafish eggs were taken from naturally matured females.
Sample preparation and assay method Embryos at the early gastrula stage were homogenized in 0.1 M Tris--HCl buffer, pH 6.8, at 0-4°C and centrifuged at 15,000g for 10rain at 2°C. The supernatant was used for determination of enzyme activity (Cahn, 1964). Seven concentrations of pyruvate spanning the range 0.1-1.0 mM were used. Apparent K~ values were calculated by the method of Lineweaver-Burk
593
594
Olga S. Klyachko and Nikolai D. Ozernyuk
using double-reciprocal plots of velocity vs substrate concentration.
Results
K m (raM)
A 0.3
Km for eggs of various fish species The effect of assay temperature on the Km value for pyruvate of LDH from embryos of fish preferring different environmental temperatures was examined. For embryos at the early gastrula stage of various species the minimum of Km as a function of assay temperature differs distinctly (Fig. 1). For rainbow trout, autumn cisco and least (Siberian) cisco eggs, the minimum Km values were 5-8°C. For zebra fish, carp and goldfish embryos, the Km values show a minimum at 25-28°C, whereas for loach they were observed at 13-16°C. For the loach eggs the effect of temperature on Km is not quite as substantial.
0.2
0.1
0
K m (raM)
04[
0.3
IAc
/ 10
I 15
I 20
Temperature (°C)
Km for embryos and larvae of rainbow trout The effect of assay temperatures on the Km values for pyruvate from the rainbow trout embryos at different stages of development, and larvae also, was studied. The embryogenesis and larval development of rainbow trout in nature proceeds at different temperatures; the early stages are at relatively low temperature, the later ones at a higher temperature. For the embryos at the early gastrula stage the Km values showed a minimum at 5-7°C, whereas for those at hatching they were 10°C (Fig. 2.). For the
I 5
Fig. 2. Temperature effect on the K m for pyruvate of LDH from extracts of rainbow trout embryos at (A) early gastrula stage, (B) hatching, and (C) skeletal muscles of larvae.
skeletal muscles of larvae the Km minimum was at 10-13°C. The question arising is whether the disclosed kinetic features of LDH from embryos and larvae of rainbow trout are related to variation of the environmental temperature. The answer was found in the experiment on the development of embryos at constant ambient temperature (5°C). It was found that in this case the position of the Km minimum was the same for the embryos at the early gastrula stage as it was for those at hatching (Fig. 3). Hence, the variation of the Km minimum, illustrated in Fig. 2, appears to be related to variation of environmental temperature.
Discussion 0.2
0.1
0
;
1'0
,'5
;5
Temperature (*(2) Fig. I. Effect of temperature on apparent K m for pyruvate of LDH from extracts of embryos at the early gastrula stage. A: rainbow trout; B: least (Siberian) cisco; C: autumn cisco; D: loach; E: goldfish; F: carp; G: zebrafish.
For various fish species the minimum Km values are correlated with the optimal temperatures in nature (Hochachka and Somero, 1973; Yancey and Somero, 1978; Coppes and Somero 1990). This conclusion is supported for the embryos of fish preferring different environmental temperatures. For the embryos of rainbow trout, autumn cisco and least (Siberian) cisco, adapted to cold habitats, the apparent Kmvalues for pyruvate of LDH were at 5-8°C, whereas for the eggs of zebrafish, carp and goldfish, preferring warm habitats, this value was at 25-28°C. For the embryos of loach, adapted to moderate temperatures, the Km minima were observed at 13-17°C (Fig. 1). These results are consistent with the data for adult fishes
LDH in fish embryos
et al., 1975; Arai and Yamazaki, 1979, 1980).
K m (raM)
0.3
0.2
595
However, in the our experiment on the development of embryos at constant temperature it was found that the position of the Km minimum was the same for the embryos at both early and later stages of embryogenesis. Thus, the variation in the Km minimum appears to be related to the variation in environmental temperature.
I
References
0.1
o
1'0
1'5
Temperature (°C) Fig. 3. Effect of temperature on the Km for pyruvate of LDH from extracts of rainbow trout embryos developing at constant temperature (5°). A: early gastrula stage; B: hatching.
preferring different environmental temperatures (Yancey and Somero, 1978; Graves and Somero, 1982; Coppes and Somero, 1990). It is known that development of rainbow trout embryos in nature proceeds at increasing temperatures. Experiments with embryos incubated at constant temperature suggest that the increase in the Km minimum position during the development is related to the increase of habitat temperature. The change in the Km minimum position may by due to changes in enzyme isoforms during embryogenesis. Indeed, the changes in LDH isozyme patterns in embryogenesis of salmonid fish were reported (Wright
Arai K. and Yamazaki F. (1979) Developmental changes of lactate dehydrogenase (LDH) isozymes in the hybrid between masu salmon (Oncorhynchus masou) and pink salmon (O. gorbuscha). Bull. Fac. Fish. Hokkaido Univ. 30, 124-128. Arai K. and Yamazaki F. (1980) Developmental genetics on the lactate dehydrogenase (LDH) isozyme during early embryogenesis of salmonids. Bull. Fac. Fish. Hokkaido Univ. 31, 223-228. Cahn R. D. (1964) Developmental changes in embryonic enzyme patterns: the effect of oxidative substrates on lactic dehydrogenase in beating chick embryonic heart cell cultures. Devl BioL 9, 327-346. Coppes Z. L. and Somero G. N. (1990) Temperatureadaptive differences between the M4-1actate dehydrogenases of stenothermal and eurythermal sciaenid fishes. J. exp. Zool. 2.54, 127-131. Graves J. E. and Somero G. N. (1982) Electrophoretic and functional enzymic evolution in four species of Eastern Pacific barracudas from different thermal environments. Evolution 36, 97-106. Hochachka P. W. and Somero G. N. (1973) Strategies of Biochemical Adaptation. W. B. Saunders, Philadelphia. Hochachka P. W. and Somero G. N. (1984) Biochemical Adaption. Princeton University Press, Princeton, NJ. Neyfakh A. A. (1959) X-ray inactivation of nuclei as a method for studying their function in the early development of fishes. J. Embryol. exp. Morph. 2, 173-192. Wright J. E., Heckman R. and Aterton L. M. (1975) Genetics and developmental analysis of LDH isozymes in trout. In Isozymes-- 3. Developmental Biology (Edited by Markert C. L.), pp. 375-401. Academic Press, New York. Yancey P. H. and Somero G. N. (1978) Temperature dependence of intracellular pH: its role in the conservation of pyruvate apparent K,, values of vertebrate lactate dehydrogenase. J. comp. Physiol. 12,5, 129-134.