- Email: [email protected]
Effect of environmental temperature upon abdominal ganglia acetylcholine hydrolase in the crayfish, Procambarus clarki girard
Comp. Biochem. Physiol., Vol. 66C, pp. 209 to 212
0306-4492/80/0701-0209502.00/0
© Pergamon Press Lid 1980. Printed in Great Britain
EFFECT OF ENVIRONMENTAL TEMPERATURE UPON ABDOMINAL GANGLIA ACETYLCHOLINE HYDROLASE IN THE CRAYFISH, P R O C A M B A R U S C L A R K I GIRARD SHIRO HORIUCHI Life Science Institute, Sophia University, Kioicho 7, Chiyoda-ku, Tokyo 102, Japan (Received 5 October 1979)
Abstract--1. Acetylcholine hydrolase (ACHE) in abdominal ganglia of the crayfish, Procambarus clarki, was studied in the animals kept in hot, warm and cold temperatures. 2. Arrhenius plots of log Vm~ vs T -t for crayfish AChE changed linearly between 15--35°C. The apparent activation energy was 2.28, 1.84and 1.52kcal/mol in the hot-, warm- and cold group of crayfish, respectively. 3. AChE affinity for AthCh (acetylthiocholine) varied with temperature, and the minimum apparent K~, value was attained at the environmental temperature of the crayfish. 4. These temperature-dependent kinetic properties of ganglia AChE were discussed in relation to the role of ACh as a neurotransmitter in Procambarus.
INTRODUCTION
Recently evidence showing that an enzyme-substrate affinity is influenced by environmental temperature has been obtained in some enzymes of poikilotherms and these findings were reviewed (Hochachka & Somero, 1973; Somero & Hochachka, 1976). As for an acetylcholine hydrolase (Acetylcholinesterase, ACHE: EC 3.1.1.7), a finding that the change of environmental temperature is compensated for by a decrease of K m (Michaelis constant) has been shown in the central nervous system of several fish (Baldwin & Hochachka, 1970; Baldwin, 1971). Acetylcholine (ACh) seems to be a neurotransmitter in crustaceans. The presence of ACh-like substances was widely indicated in the ganglia of eleven species of crustaceans (Welsh, 1961). However, no conclusive evidence was obtained to support the existence of cholinergic transmission in the crustacean nervous system (Wiersma, 1961). Recently the existence of ACh and its role in transmission were demonstrated in prawn, Palaemonetes varians (Veldsema-Currie, 1973) and lobster, Homarus americanus (Hildebrand et al., 1974). It was previously reported that crayfish LDH from the abdominal flexor muscle showed a regulatory function in the change of environmental temperature (Narita & Horiuchi, 1979). In the present investigation, an attempt has been made to clarify the kinetic properties of abdominal ganglia AChE of the crayfish exposed to different temperatures, for the purpose of elucidating the biochemical mechanism adaptive to temperature in crustaceans. MATERIALS A N D METHODS
Exposure of crayfish to temperature
Freshwater crayfish, Procambarus clarki Girard, were purchased from Nippon Bio-Supplies Centre (Tokyo), and 209
kept in the laboratory. The animals were separated into 3 groups: a hot group of crayfish kept at 30 ± 2°C, a warm group at 15 ± 2°C, and a cold group at 3 _+ 2°C. The procedures in detail were previously described by Narita & Horiuchi (1979). Preparation of the enzyme Crayfish abdominal ganglia (ca. 0.02 g per one animal) were obtained by cutting the abdominal flexor muscle carefuily with anatomical scissors. After washing in chilled 10 mM Tris-HCl buffer (pH 7.2), the ganglia were homogenized with a glass-glass homogenizer in the above buffer at 4°C, then centrifuged at 1000g for I0 min and the supernatant was then used as a source of ACHE. Assays AChE activity was assayed photometrically according to the method of Ellman et al. (1961), using acetylthiocholine iodide (AthCh) as substrate. The reaction mixtures contained 10 mM DTNB (5: 5-dithiobis-nitrobenzoic acid) and 0.I M Tris-HCl (pH 7.4). The enzyme samples were preincubated at 25°C for 10rain, and then the AthCh was added. The optical density of yellow colour produced from thiocholine was measured by the Hitachi model 124 spectrophotometer at 412 nm for the first 10-20 min of the reaction. The specific activity was expressed in pxnols AthCh hydrolyzed/min/mg protein. Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard. AChE activity was completely inhibited by eserine of more than 100/zM concentration.
RESULTS Inhibition by substrate
Figure 1 shows the specific activity of AChE with varying AthCh concentrations at pH 7.5 and 25°C. The abdominal ganglia AChE of the crayfish displayed no marked substrate inhibition except in the cold group of crayfish exposed to low temperature.
210
SHIRO HORIUCHI 2.0
u
"~ ~
e e 1'5
0
1
2
3
Concentration of AthCh
z,
:t.
(mM)
Fig. I. Effect of substrate concentration on abdominal ganglia AChE of crayfish kept at different temperature. Crayfish kept in hot (O O), warm (A A) and cold ([2 El) environments were employed. Reaction mixtures contained 10raM DTNB, 0.1 M Tris-HCl (pH7.4), the enzyme samples and AthCh, and AChE activity was assayed at pH 7.5 and 25°C. The experimental conditions in detail were described in Materials and Methods.
Optimal pH As with the effect of pH on ganglia AChE activity, as shown in Fig. 2, the same effect was observed in all three groups of animals. Optimal pHs were found approx at pH 8.0. Figure 3 shows the affinity of substrate to ganglia AChE at different assay temperatures, calculated from Lineweaver-Burk plots. The apparent K s increased at temperatures above and below that of the environment for the crayfish. Thus, the minimum K s for abdominal ganglia AChE of the crayfish in the environment at 2-5°C occurred at about 4°C of the assay temperature and for the crayfish kept in 14-17°C at about 17°C and for the crayfish kept in 28-31°C at about 30°C. For all three environmental temperatures, as shown in Fig. 3, the apparent K~ values were
i
9
pH Fig. 2. Effect of pH on abdominal ganglia AChE from crayfish kept at different temperature. Hot group (O O), warm group (A A), and cold groups of crayfish (D [2). approx the same, i.e. K~, at 4°C, Km at 17°C and K m at 30°C were 1.9x 10 -4 , 2 . 4 x 10 -4 and 1.8 x 10 -4 M respectively. The rate of substrate hydrolysis was also determined. The rate of AthCh hydrolysis at assay temperature was divided by the rate at minimum Km temperature. As shown in Table I, the rate of hydrolysis was close to 1.00 at assay temperatures approaching the minimum Km in all three crayfish groups.
Arrhenius plots Arrhenius plots of log Vmaxvs T-1 for .abdominal ganglia AChE from crayfish are shown in Fig. 4. In all three crayfish groups kept in different temperatures, the log Vmaxincreased linearly with a temperature of 15--35°C. The critical point found in the case of the
10
~o6 x
EL
10
20 30 Temperature
40
50
(°C)
Fig. 3. Effect of environmental temperature on apparent K , of AthCh for AChE from crayfish abdominal ganglia. Hot group (O O), warm group-(A A), and cold group crayfish (D rl). Assay temperature is shown on abscissa.
211
Temperature effects in the crayfish
Temperature (*C) --//,
~
30 e
20 i
0.5
0 .-I
)//t
I
I
3.2
3.3
3.4
3.5
I~T x 10 3 Fig. 4. Arrhenius plot of abdominal ganglia AChE of crayfish kept in different temperatures. Hot group (O O), warm group (A A), and cold group crayfish (13 [:3). abdominal flexor muscle L D H in crayfish (Narita & Horiuchi, 1979) was not observed in abdominal ganglia ACHE. The apparent activation energy (Ea), calculated from Arrhenius plots was 2.28 kcal/mol in the hot group, 1.87kcal/mol in the warm group and 1.52/mol in the cold group crayfish, respectively.
DISCUSSION It was demonstrated that the abdominal ganglia AChE activity in Procambarus was compensated for by the decrease of the K . value for the change of environmental temperature, and that the minimum K . value approximately coincided with the habitat of the animal (Fig. 3). The physiological significance of the K.-temperature relationship was explained at temperatures above that at which Km is at a minimum. An increase in the velocity of the enzyme reaction due to a rise in temperature is presumably neutralized by a decrease in the enzyme-substrate affinity (Baldwin & Hochachka,
1970). This type of temperature effect was observed in the rate of the AthCh hydrolysis (Table 1). It was also shown that the increase in the apparent Km value indicated negative modulation below the environmental temperature (Narita & Horiuchi, 1979). It is reported in choline acetyltransferase from the goldfish brain that the amount of active enzymes does not change with temperature acclimation, but the brain is able to synthesize a new and more active enzyme at higher temperature (Hebb et al., 1969). Baldwin & Hochachka (1970) demonstrated electrophoretically that the production of different isozymes occurs seasonally in rainbow trout brain ACHE. It was also shown that the activation energy of ganglia AChE calculated from the Arrhenius plot became smaller in the crayfish exposed respectively to high, warm, and low temperature, i.e. 2.28, 1.84 and 1.52 kcal/mol (Fig. 4). A linear relationship was observed between Vmax and T - 1 but a critical point, which was remarkable in abdominal muscle LDH in Procambarus (Narita & Horiuchi, 1979), was not seen.
Table 1. Effect of temperature upon the rate of AthCh hydrolysis at K,, concentration of substrate for crayfish abdominal ganglia AChE Crayfish
Temp. at minimum Km (°C)
Cold group
4
Warm group
17
Hot group
30
Assay temp. (°C)
Rate of hydrolysis*
4 5 10 15 i0 15 17
1.00 1.10 1.12 1.21 0.75 1.00 1.00
20
1.11
25 25 30 35 42
1.23 0.80 1.00 1.10 1.24
* Rate of hydrolysis at assay temperature was divided by the rate at minimum Km temperature. The average of 3-5 determinations.
212
SHIRO HORIUCHI
BULLOCKT. H. (1955) Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30, 311-342. CP.AWSHAWL. I. (1974) Temperature selection and activity in the crayfish, Orconectes immunis. J. comp. Physiol. 95, 315-322. ELLMANG. L., COURTNEYK. D., ANDRESV. JR • FEARHERSTONER. M. (1961) A new and rapid colorimetric determination of acetyicholinesterase activity. Biochem. Pharmae. 7, 88-95. HEBBC., MORRISD. & SMITHM. W. (1969) Choline acetyltransferase activity in the brain of goldfish acclimated to different temperatures. Comp. Biochem. Physiol. 28, 29-36. HILDEBRANDJ. G., TOWNSELJ. G. & KRAVITZE. A. (1974) Distribution of acetylcholine, choline, choline acetyltransferase and acetylcholinesterase in regions and single identified axons of the lobster nervous system. J. Neurochem. 23, 951-963. HOCHACHKAP. W. & SOMEROG. N. (1973) Strategies of Biochemical Adaptation. Saunders, Philadelphia. LowRY O. H., ROSEBROUOHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent, d. biol. Chem. 193, 265-275. NARITAJ. & HORIUCHIS. (1979) Effect of environmental temperature upon muscle lactate dehydrogenase in the crayfish, Procambarus clarki Girard. Comp. Biochem. Physiol. 64, 279-283. SOMEROG. N. & HOCHACHKAP. W. (1976) Biochemical Acknowledgements--The author would like to thank Adaptations to Temperature. In Adaptation to EnvironProfessor Daniel McCoy, S. J. Sophia University for ment (Edited by NEWELL R. C.), pp. 125-190. Butterreviewing this manuscript and Mr N. Takanashi for his worths, London. technical assistance. VELDSEMA-CuRRIER. D. (1973) Some components of the cholinergic system in the prawn Palaemonetes varians (Leach). Biochem. J. 135, 673-682. WELSHJ. H. (1961) Neurohumours and neurosecretion. In REFERENCES The Physiology of the Crustacea, Vol. 2 (Edited by WATERMANT. H.), pp. 281-311. Academic Press, New BALDWINJ. & HOCHACHKAP. W. (1970) Functional signifiYork. cance of isozymes in thermal acclimation. AcetylcholiWlERSMAC. A. G. (1961) Reflexes and the central nervous nesterase from trout brain. Biochem. J. 116, 883-887. system. In The Physiology of the Crustacea, eel. 2 BALDWINJ. (1971) Adaptation of enzymes to temperature: (Edited by WATERMANT. H.). pp. 241-279. Academic acetylcholinesterase in the central nervous system of Press, New York. fishes. Comp. Biochem. Physiol. 40, 181-187.
As for the metabolic response to temperature change of an European crayfish, Bullock (1955) reported that 5°C-acclimated crayfish achieved a new high metabolic level, although the crayfish responded slowly to a temperature rise compared with trout. Crayfish are known to be sensitive to environmental temperature (Crawshaw, 1974). Thus, abdominal ganglia AChE presumably will play a role in the metabolism of thermoreception in Procambarus. High AChE activity comparable to mammalian AChE was found in the nervous tissue of the prawn, Palaemonetes varians. The wide distribution of AChE in the brain, ventral cord, abdominal muscle and other parts is thought to support the function of ACh as sensory transmitter in P. varians (Veldsema-Currie, 1973). The presence of ACh and the high activity of AChE were also reported in sensory axons from abdominal muscle of the lobster, Homarus americanus. Uniform distribution of ACh, AChE and choline acetyltransferase in nerve and muscle of the lobster strengthens the proposal that ACh is the sensory neurotransmitter in the lobster (Hildebrand et al., 1974). In crayfish also, it is probable that the AChAChE system is involved in the transmission of the regulation of environmental temperature changes.
0306-4492/80/0701-0209502.00/0
© Pergamon Press Lid 1980. Printed in Great Britain
EFFECT OF ENVIRONMENTAL TEMPERATURE UPON ABDOMINAL GANGLIA ACETYLCHOLINE HYDROLASE IN THE CRAYFISH, P R O C A M B A R U S C L A R K I GIRARD SHIRO HORIUCHI Life Science Institute, Sophia University, Kioicho 7, Chiyoda-ku, Tokyo 102, Japan (Received 5 October 1979)
Abstract--1. Acetylcholine hydrolase (ACHE) in abdominal ganglia of the crayfish, Procambarus clarki, was studied in the animals kept in hot, warm and cold temperatures. 2. Arrhenius plots of log Vm~ vs T -t for crayfish AChE changed linearly between 15--35°C. The apparent activation energy was 2.28, 1.84and 1.52kcal/mol in the hot-, warm- and cold group of crayfish, respectively. 3. AChE affinity for AthCh (acetylthiocholine) varied with temperature, and the minimum apparent K~, value was attained at the environmental temperature of the crayfish. 4. These temperature-dependent kinetic properties of ganglia AChE were discussed in relation to the role of ACh as a neurotransmitter in Procambarus.
INTRODUCTION
Recently evidence showing that an enzyme-substrate affinity is influenced by environmental temperature has been obtained in some enzymes of poikilotherms and these findings were reviewed (Hochachka & Somero, 1973; Somero & Hochachka, 1976). As for an acetylcholine hydrolase (Acetylcholinesterase, ACHE: EC 3.1.1.7), a finding that the change of environmental temperature is compensated for by a decrease of K m (Michaelis constant) has been shown in the central nervous system of several fish (Baldwin & Hochachka, 1970; Baldwin, 1971). Acetylcholine (ACh) seems to be a neurotransmitter in crustaceans. The presence of ACh-like substances was widely indicated in the ganglia of eleven species of crustaceans (Welsh, 1961). However, no conclusive evidence was obtained to support the existence of cholinergic transmission in the crustacean nervous system (Wiersma, 1961). Recently the existence of ACh and its role in transmission were demonstrated in prawn, Palaemonetes varians (Veldsema-Currie, 1973) and lobster, Homarus americanus (Hildebrand et al., 1974). It was previously reported that crayfish LDH from the abdominal flexor muscle showed a regulatory function in the change of environmental temperature (Narita & Horiuchi, 1979). In the present investigation, an attempt has been made to clarify the kinetic properties of abdominal ganglia AChE of the crayfish exposed to different temperatures, for the purpose of elucidating the biochemical mechanism adaptive to temperature in crustaceans. MATERIALS A N D METHODS
Exposure of crayfish to temperature
Freshwater crayfish, Procambarus clarki Girard, were purchased from Nippon Bio-Supplies Centre (Tokyo), and 209
kept in the laboratory. The animals were separated into 3 groups: a hot group of crayfish kept at 30 ± 2°C, a warm group at 15 ± 2°C, and a cold group at 3 _+ 2°C. The procedures in detail were previously described by Narita & Horiuchi (1979). Preparation of the enzyme Crayfish abdominal ganglia (ca. 0.02 g per one animal) were obtained by cutting the abdominal flexor muscle carefuily with anatomical scissors. After washing in chilled 10 mM Tris-HCl buffer (pH 7.2), the ganglia were homogenized with a glass-glass homogenizer in the above buffer at 4°C, then centrifuged at 1000g for I0 min and the supernatant was then used as a source of ACHE. Assays AChE activity was assayed photometrically according to the method of Ellman et al. (1961), using acetylthiocholine iodide (AthCh) as substrate. The reaction mixtures contained 10 mM DTNB (5: 5-dithiobis-nitrobenzoic acid) and 0.I M Tris-HCl (pH 7.4). The enzyme samples were preincubated at 25°C for 10rain, and then the AthCh was added. The optical density of yellow colour produced from thiocholine was measured by the Hitachi model 124 spectrophotometer at 412 nm for the first 10-20 min of the reaction. The specific activity was expressed in pxnols AthCh hydrolyzed/min/mg protein. Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard. AChE activity was completely inhibited by eserine of more than 100/zM concentration.
RESULTS Inhibition by substrate
Figure 1 shows the specific activity of AChE with varying AthCh concentrations at pH 7.5 and 25°C. The abdominal ganglia AChE of the crayfish displayed no marked substrate inhibition except in the cold group of crayfish exposed to low temperature.
210
SHIRO HORIUCHI 2.0
u
"~ ~
e e 1'5
0
1
2
3
Concentration of AthCh
z,
:t.
(mM)
Fig. I. Effect of substrate concentration on abdominal ganglia AChE of crayfish kept at different temperature. Crayfish kept in hot (O O), warm (A A) and cold ([2 El) environments were employed. Reaction mixtures contained 10raM DTNB, 0.1 M Tris-HCl (pH7.4), the enzyme samples and AthCh, and AChE activity was assayed at pH 7.5 and 25°C. The experimental conditions in detail were described in Materials and Methods.
Optimal pH As with the effect of pH on ganglia AChE activity, as shown in Fig. 2, the same effect was observed in all three groups of animals. Optimal pHs were found approx at pH 8.0. Figure 3 shows the affinity of substrate to ganglia AChE at different assay temperatures, calculated from Lineweaver-Burk plots. The apparent K s increased at temperatures above and below that of the environment for the crayfish. Thus, the minimum K s for abdominal ganglia AChE of the crayfish in the environment at 2-5°C occurred at about 4°C of the assay temperature and for the crayfish kept in 14-17°C at about 17°C and for the crayfish kept in 28-31°C at about 30°C. For all three environmental temperatures, as shown in Fig. 3, the apparent K~ values were
i
9
pH Fig. 2. Effect of pH on abdominal ganglia AChE from crayfish kept at different temperature. Hot group (O O), warm group (A A), and cold groups of crayfish (D [2). approx the same, i.e. K~, at 4°C, Km at 17°C and K m at 30°C were 1.9x 10 -4 , 2 . 4 x 10 -4 and 1.8 x 10 -4 M respectively. The rate of substrate hydrolysis was also determined. The rate of AthCh hydrolysis at assay temperature was divided by the rate at minimum Km temperature. As shown in Table I, the rate of hydrolysis was close to 1.00 at assay temperatures approaching the minimum Km in all three crayfish groups.
Arrhenius plots Arrhenius plots of log Vmaxvs T-1 for .abdominal ganglia AChE from crayfish are shown in Fig. 4. In all three crayfish groups kept in different temperatures, the log Vmaxincreased linearly with a temperature of 15--35°C. The critical point found in the case of the
10
~o6 x
EL
10
20 30 Temperature
40
50
(°C)
Fig. 3. Effect of environmental temperature on apparent K , of AthCh for AChE from crayfish abdominal ganglia. Hot group (O O), warm group-(A A), and cold group crayfish (D rl). Assay temperature is shown on abscissa.
211
Temperature effects in the crayfish
Temperature (*C) --//,
~
30 e
20 i
0.5
0 .-I
)//t
I
I
3.2
3.3
3.4
3.5
I~T x 10 3 Fig. 4. Arrhenius plot of abdominal ganglia AChE of crayfish kept in different temperatures. Hot group (O O), warm group (A A), and cold group crayfish (13 [:3). abdominal flexor muscle L D H in crayfish (Narita & Horiuchi, 1979) was not observed in abdominal ganglia ACHE. The apparent activation energy (Ea), calculated from Arrhenius plots was 2.28 kcal/mol in the hot group, 1.87kcal/mol in the warm group and 1.52/mol in the cold group crayfish, respectively.
DISCUSSION It was demonstrated that the abdominal ganglia AChE activity in Procambarus was compensated for by the decrease of the K . value for the change of environmental temperature, and that the minimum K . value approximately coincided with the habitat of the animal (Fig. 3). The physiological significance of the K.-temperature relationship was explained at temperatures above that at which Km is at a minimum. An increase in the velocity of the enzyme reaction due to a rise in temperature is presumably neutralized by a decrease in the enzyme-substrate affinity (Baldwin & Hochachka,
1970). This type of temperature effect was observed in the rate of the AthCh hydrolysis (Table 1). It was also shown that the increase in the apparent Km value indicated negative modulation below the environmental temperature (Narita & Horiuchi, 1979). It is reported in choline acetyltransferase from the goldfish brain that the amount of active enzymes does not change with temperature acclimation, but the brain is able to synthesize a new and more active enzyme at higher temperature (Hebb et al., 1969). Baldwin & Hochachka (1970) demonstrated electrophoretically that the production of different isozymes occurs seasonally in rainbow trout brain ACHE. It was also shown that the activation energy of ganglia AChE calculated from the Arrhenius plot became smaller in the crayfish exposed respectively to high, warm, and low temperature, i.e. 2.28, 1.84 and 1.52 kcal/mol (Fig. 4). A linear relationship was observed between Vmax and T - 1 but a critical point, which was remarkable in abdominal muscle LDH in Procambarus (Narita & Horiuchi, 1979), was not seen.
Table 1. Effect of temperature upon the rate of AthCh hydrolysis at K,, concentration of substrate for crayfish abdominal ganglia AChE Crayfish
Temp. at minimum Km (°C)
Cold group
4
Warm group
17
Hot group
30
Assay temp. (°C)
Rate of hydrolysis*
4 5 10 15 i0 15 17
1.00 1.10 1.12 1.21 0.75 1.00 1.00
20
1.11
25 25 30 35 42
1.23 0.80 1.00 1.10 1.24
* Rate of hydrolysis at assay temperature was divided by the rate at minimum Km temperature. The average of 3-5 determinations.
212
SHIRO HORIUCHI
BULLOCKT. H. (1955) Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30, 311-342. CP.AWSHAWL. I. (1974) Temperature selection and activity in the crayfish, Orconectes immunis. J. comp. Physiol. 95, 315-322. ELLMANG. L., COURTNEYK. D., ANDRESV. JR • FEARHERSTONER. M. (1961) A new and rapid colorimetric determination of acetyicholinesterase activity. Biochem. Pharmae. 7, 88-95. HEBBC., MORRISD. & SMITHM. W. (1969) Choline acetyltransferase activity in the brain of goldfish acclimated to different temperatures. Comp. Biochem. Physiol. 28, 29-36. HILDEBRANDJ. G., TOWNSELJ. G. & KRAVITZE. A. (1974) Distribution of acetylcholine, choline, choline acetyltransferase and acetylcholinesterase in regions and single identified axons of the lobster nervous system. J. Neurochem. 23, 951-963. HOCHACHKAP. W. & SOMEROG. N. (1973) Strategies of Biochemical Adaptation. Saunders, Philadelphia. LowRY O. H., ROSEBROUOHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent, d. biol. Chem. 193, 265-275. NARITAJ. & HORIUCHIS. (1979) Effect of environmental temperature upon muscle lactate dehydrogenase in the crayfish, Procambarus clarki Girard. Comp. Biochem. Physiol. 64, 279-283. SOMEROG. N. & HOCHACHKAP. W. (1976) Biochemical Acknowledgements--The author would like to thank Adaptations to Temperature. In Adaptation to EnvironProfessor Daniel McCoy, S. J. Sophia University for ment (Edited by NEWELL R. C.), pp. 125-190. Butterreviewing this manuscript and Mr N. Takanashi for his worths, London. technical assistance. VELDSEMA-CuRRIER. D. (1973) Some components of the cholinergic system in the prawn Palaemonetes varians (Leach). Biochem. J. 135, 673-682. WELSHJ. H. (1961) Neurohumours and neurosecretion. In REFERENCES The Physiology of the Crustacea, Vol. 2 (Edited by WATERMANT. H.), pp. 281-311. Academic Press, New BALDWINJ. & HOCHACHKAP. W. (1970) Functional signifiYork. cance of isozymes in thermal acclimation. AcetylcholiWlERSMAC. A. G. (1961) Reflexes and the central nervous nesterase from trout brain. Biochem. J. 116, 883-887. system. In The Physiology of the Crustacea, eel. 2 BALDWINJ. (1971) Adaptation of enzymes to temperature: (Edited by WATERMANT. H.). pp. 241-279. Academic acetylcholinesterase in the central nervous system of Press, New York. fishes. Comp. Biochem. Physiol. 40, 181-187.
As for the metabolic response to temperature change of an European crayfish, Bullock (1955) reported that 5°C-acclimated crayfish achieved a new high metabolic level, although the crayfish responded slowly to a temperature rise compared with trout. Crayfish are known to be sensitive to environmental temperature (Crawshaw, 1974). Thus, abdominal ganglia AChE presumably will play a role in the metabolism of thermoreception in Procambarus. High AChE activity comparable to mammalian AChE was found in the nervous tissue of the prawn, Palaemonetes varians. The wide distribution of AChE in the brain, ventral cord, abdominal muscle and other parts is thought to support the function of ACh as sensory transmitter in P. varians (Veldsema-Currie, 1973). The presence of ACh and the high activity of AChE were also reported in sensory axons from abdominal muscle of the lobster, Homarus americanus. Uniform distribution of ACh, AChE and choline acetyltransferase in nerve and muscle of the lobster strengthens the proposal that ACh is the sensory neurotransmitter in the lobster (Hildebrand et al., 1974). In crayfish also, it is probable that the AChAChE system is involved in the transmission of the regulation of environmental temperature changes.