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A note on studies of in vitro protein synthesis in temperature acclimated teleost tissues
Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 557 to 559. Pergamon Press. Printed in Great Britain
A NOTE ON STUDIES OF IN VITRO PROTEIN SYNTHESIS IN TEMPERATURE ACCLIMATED TELEOST TISSUES TERRANCE G. OWEN Chemistry Dept., Simon Fraser University, Burnaby, B.C. Canada V5A 1S6 (Received 31 July 1974)
Abstract--l. Both the inulin space and the rate of leucine incorporation into protein were 150% greater in vitro in livers from warm acclimated rainbow trout compared to those from cold acclimated trout. 2. Temperature acclimation had no effect on the uptake of leucine into the free amino acid pool. 3. Since the precursor pool of amino acids for protein synthesis appears to be extracellular, alteration of the extracellular space by temperature shock is believed to have affected protein synthesis rates by interfering with the transport of amino acids from this pool to the site of protein synthesis.
homogenized in 2 ml of 0.5 N NaOH which was expected to halt subcellular processes and enhance protein solubilization. The debris was removed by centrifuging at 2000 g for 10 min and duplicate 0.1-0.4 ml samples of the supernatant were taken for biuret determination of protein while additional 0"5 ml samples were added to 1 ml of 10% trichloroacetic acid. The trichloroacetic acid-protein mixtures were centrifuged after standing overnight and 0'4 ml aliquots of the supernatant were dissolved in scintillation vials with 10 ml of Aquasol (New England Nuclear). The precipitates were washed twice with 5% trichloroacetic acid and twice with 95% ethanol, solubilized by the addition of 0'05 ml water and 0.5 ml of Eastman Tissue Solubilizer and transferred quantitatively into vials with 10 ml of toluene containing 6 g of PPO and 0-5 of POPOP per liter. All samples were counted using Program 8 of a Nuclear Chicago Isocap 300 scintillation counter. The results of the zero min samples were subtracted from the subsequent samples of each experiment to correct for background counts and radioactivity taken up by disrupted cells. All means were compared by the Student's t-test, all variations represent the S.E.M. and in all cases, N = 6.
INTRODUCTION
IT IS well k n o w n that the rates of m a n y metabolic processes are e n h a n c e d during cold acclimation in poikilotherms to compensate for the lower levels of thermal energy available (Fry & Hochachka, 1970). This includes protein synthesis in teleost fishes in vivo (Das, 1967; D e a n & Berlin, 1969; Haschemeyer, 1969b, 1973; Haschemeyer & Persell, 1973; M o r r i s & Smith, 1967; Smith & Morris, 1966) a n d in cell free extracts (Haschemeyer, 1969b). However, in the present study of in vitro protein synthesis in diced livers from temperature acclimated trout, the opposite effect was observed. A possible explanation for this a n o m a l o u s result is provided in this report. MATERIALS AND METHODS Experimental animals The trout, Salma .qairdneri (approx. 100g) were acclimated to 5 and 15°C in 4001 tanks for at least 2 weeks and fed once daily with Clark's rainbow trout pellets. The light regimen was 12 hr light: 12 hr darkness and water was supplied at 1"51/min.
RESULTS
Tissue preparation The fish were stunned, the livers were excised and a sample of the liver was retained for soluble protein determination by the biuret method. The remainder was diced into pieces about 1 mm 3, rinsed 12 times with 5 ml aliquots of rainbow trout saline (Stokes & Fromm, 1964) also containing 5 mM glucose, streptomycin sulphate (100 mg/ml) and penicillin (50 units/ml) and saturated with 95~o 02 : 5~o CO2 (termed the basic medium) and preincubated in 20 ml of this same medium for 1 hr. All incubations contained one liver and were conducted at 10°C using a shaker bath and continuous gassing with the 02 :CO2 mixture.
The up .take of labelled leucine into the trichloroacetic acid soluble fraction was identical for w a r m a n d cold livers (Fig. 1).
Incorporation studies The liver cubes were retrieved on cheesecloth and added to 10 ml of the basic medium containing 0.1 mM leucine, 1.87 #C/ml L-[4,5-H a] leucine (specific activity-29.8 C/mM) and 0.35 #C/ml [carboxyl-C 14] inulin (specific activity2.68 mC/g), both from International Chemical and Nuclear. Samples were withdrawn with a pipet at 0, 20, 40, 60, 90, 120, 150 and 180 min and excess medium was removed by vacuum filtration on glass fiber filters. The samples were rinsed 7 times with 5 ml aliquots of basic medium and then 557
24-5*
× =_
16--
-o
I
I
I
2
P
5
hr
Fig. 1. In vitro uptake at 10°C of L-[4,5-H a] leucine into trichloroaeetic acid soluble material by liver cubes of rainbow trout acclimated to 5 and 15°C. For each point N = 6.
558
TERRAN(?E G . OWEN
i
6-
~!~"1
o.....-~o
I I mo
t I
•
min/mg protein for the warm and cold livers, respectively. DISCUSSION
×
4
I
I
I
2
I
3
hr"
Fig. 2. In vitro uptake at 10°C of [carboxyl-C ~4] inulin by liver cubes of rainbow trout acclimated to 5 and 15°C. For each point N = 6. Vertical bars represent + S.E.
Inulin saturation required about 120min and the warm livers took up 150% more inulin than the cold livers (Fig. 2). The values for inulin uptake were significantly different (P < 0.05) for all sampling times after 40 rain. Comparisons based on the protein determinations were considered to be valid since no significant difference in the protein content per unit wet weight was found between the warm and cold livers. The extracellular space was calculated according to Reiger & Kafatos (1971) and found to be 0'775 + 0-083 and 0"505 + 0'082 p.l/mg protein for warm and cold livers, respectively. These values were significantly different (P < 0.05) and accepted as a true representation of the comparative extracellular space since the H3/ C 14 ratios of the rinses of the labelled tissues were not significantly different from that of the incubation medium. This indicates that inulin was not bound by the tissues (Reiger & Kafatos, 1971). The rate of incorporation of labelled leucine into total protein was 150% greater in the warm livers than the cold livers (Fig. 3). The rates were significantly different (P < 0-01) and were 46 and 31 dis/min per 9 15o
'O
o.
"o
I
2
5
hr
Fig. 3. In vitro incorporation at 10°C of L-[-4,5,H3] leucine into trichloroacetic acid insoluble material by liver cubes of rainbow trout acclimated to 5 and 15°C.The slopes are significantly different (P < 0-01). For each point N = 6.
The greater rate of protein synthesis observed in the warm livers in this investigation seems unusual since other workers have consistently found that the rates of this process are enhanced by cold acclimation (Das, 1967; Dean & Berlin, 1969, Haschemeyer, 1969a,b, 1973; Hascbemeyer & Persell, 1973; Morris & Smith, 1967; Smith & Morris, 1966). However, all of these studies were conducted in vivo with one exception (Haschemeyer, 1969a) in which cell-free extracts were used. Consequently, the different results obtained in the present study are believed to be due to the use ol diced livers, a method that has not been previously exploited for temperature acclimation studies. Although potentially useful in that it is more convenient and offers increased control over experimental conditions compared to in civo studies and still allows examination of the responses of intact tissues, this approach seems to present certain pitfalls when applied to studies of temperature acclimation. This becomes obvious when the anomalous protein synthesis rates are considered. The basis for this reversal of expected results does not appear to reside in the rate of penetration of labelled leucine into the total amino acid pool since these rates are essentially identical (assuming that the specific radioactivities are the same). However, the difference in the rates of protein synthesis coincides exactly to the difference in the extracellular spaces. Also, the precursor pool for protein synthesis appears to be extracellular or in rapid equilibrium with the extracellular pool since the protein synthesis plots are straight lines that pass through the origin (Hider et al., 1969, 1971 ; Kipnis et al., 1961 ; Rosenberg et al., 1963; VanVerooij et al., 1972). Although this conclusion would be supported more conclusively with data from the use of two different isotopic forms of the same amino acid (Alpers & Thier, 1972; Hider et al., 1969, 1971; VanVerooij et al., 1972)it seems valid since there is usually a time lag before a maximum rate of protein synthesis is attained if the precursor pool is intracellular (Alpers & Thier, 1972; Morgan et al., 1971; Reiger & Kafatos, 1971 ; Rosenberg et al., 1963). There may be a direct relationship then, between the volume of the extracellular space and the rate of protein synthesis. The reason for this is not known but if one accepts the hypothesis of Hider et al. (1969) that the external amino acids enter the cellular membrane via "carriers" and are transported directly to the site of protein synthesis without mixing with the intracellular pool, then it seems possible that a reduction of extracellular space could affect the rate of protein synthesis by reducing the number of functional carriers and the amount of amino acids available to the remaining carriers. Since other workers have observed significant, transitory alterations in the extracellular space in teleost tissues as a result of abrupt temperature changes (Heinicke & Houston, 1965; Hickman et al., 1964) it seems particularly possible that such a reduction may have occurred in the cold livers in this study. If so, it is concluded that this paper has illustrated that failure to consider the effects of abrupt temperature changes on the extracellular space in such
Protein synthesis in teleosts temperature acclimation studies could lead to erroneous conclusions. Also, it seems that the in vitro methods described m a y create more problems than they solve despite the theoretical advantages. Obviously, more refinement is required. Acknowledoements--This research was conducted at the University of British Columbia, Vancouver, B.C., Canada and was supported by N. C. R. of Canada grant No. 67-3678 to the author's supervisor of doctoral studies, Dr. P. W. Hochachka of the Zoology Dept., and F. R. B. of Canada grant No. 65-1848.
REFERENCES
ALPERS D. H. & THIER S. O. (1972) Role of the free amino acid pool of the intestine in protein synthesis. Biochim. biophys. Acta 262, 535-545. DAS A. B. (1967) Biochemical changes in tissues of goldfish acclimated to high and low temperatures---II. Synthesis of protein and RNA of subcellular fractions and tissue composition. Comp. Biochem. Physiol. 21,469-485. DEAN J. M. & BERLIN J. D. (1969) Alterations in tiepatocyte function of thermally acclimated rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 29, 307-312. FRY F. E. J. & HOCHACHKA, P. W. (1970) Fish. In Comparative Physiology of Thermoregulation (Edited by WHITTOW G. C.), Vol. I, pp. 79-134. Academic Press, New York. HASCHEMEYERA. E. V. (1969a) Studies on the control of protein synthesis in low temperature acclimation. Comp. Bigchem. Physiol. 28, 535-552. HASCHEMEYERA. E. V. (1969b) Rates of polypeptide chain assembly in liver in vivo: relation to the mechanism of temperature acclimation in Opsanus tau. Proc. natn. Acad. Sci. U.S.A. 62, 128-135. HASCHEMEYERA. E. V. (1973) Kinetic analysis of synthesis and secretion of plasma protein in a marine teleost. J. Biol. Chem. 248, 1643-1649. HASCHE~WR A. E. V. & PERSELL R. (1973) Kinetic studies on amino acid uptake and protein synthesis in liver of temperature acclimated toadfish. Biol. Bull. 145, 472-481.
559
HEINICKE E. A. & HOUSTON A. G. (1965) Effect of thermal acclimation and sublethal heat shock upon ionic regulation in the goldfish, Carassius auratus L. d. Fish Res. Bd Can. 22, 1455-1476. HICKMANC. P. JR., McNABB R. A., NELSON J. S., VANBREEMAN E. D. & CO~FORT D. (1964) Effect of cold acclimation on electrolyte distribution in rainbow trout (Salmo #airdneri). Can. J. Zool. 42, 577-597. HIDER R. C., FERN E. B. & LONDON D. R. (1969) Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats. Biochem. J. 114, 171-178. HIDER R. C., FERN E. B. & LONDON D. R. (1971) Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis. Biochem. J. 121, 817-827. KIPNIS D. M., REISS E. & HELMREICH E. (1961) Functional heterogeneity of the intracellular amino acid pool in mammalian cells. Biochim. biophys. Acta 51,519-524. MORGAN H. E., EARL D. C. N., BROADUS A., WOLPERT E. B., GIGER K. E. & JEFFERSON L. S. (1971) Regulation of protein synthesis in heart muscle--l. Effect of amino acid levels on protein synthesis. J. Biol. Chem. 246, 2152-2162. MORRIS D. & SMITH M. W. (1967) Protein synthesis in the intestine of goldfish acclimatized to different temperatures. Biochem. J. 102, 648-653. RElGER J. C. & KAFATOS F. C. (1971) Microtechnique for determining the specific activity of radioactive intracellular leucine and applications to in vivo studies of protein synthesis. J. Biol. Chem. 246, 6480-6488. ROSENBERGL. E., BERMANM. & SEGAL S. (1963) Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney cortex slices. Biochim. biophys. Acta 71, 664-675. SMITH M. W. & MORRISD. (1966) Temperature acclimatization and protein synthesis in the goldfish mucosa. Experientia 22, 678-679. STORES R. M. & FROMM P. O. (1964) Glucose absorption and metabolism by the gut of rainbow trout. Comp. Bigchem. Physiol. 13, 53-69. VANVEROOIJW. J., POORT C., KRAMER M. F. & JANSEN M. T. (1972) Relationship between extracellular amino acids and protein synthesis in vitro in the rat pancreas. Eur. J. Biochem. 30, 427-433. Key Word Index--Extracellular space; protein synthesis; temperature acclimation.
A NOTE ON STUDIES OF IN VITRO PROTEIN SYNTHESIS IN TEMPERATURE ACCLIMATED TELEOST TISSUES TERRANCE G. OWEN Chemistry Dept., Simon Fraser University, Burnaby, B.C. Canada V5A 1S6 (Received 31 July 1974)
Abstract--l. Both the inulin space and the rate of leucine incorporation into protein were 150% greater in vitro in livers from warm acclimated rainbow trout compared to those from cold acclimated trout. 2. Temperature acclimation had no effect on the uptake of leucine into the free amino acid pool. 3. Since the precursor pool of amino acids for protein synthesis appears to be extracellular, alteration of the extracellular space by temperature shock is believed to have affected protein synthesis rates by interfering with the transport of amino acids from this pool to the site of protein synthesis.
homogenized in 2 ml of 0.5 N NaOH which was expected to halt subcellular processes and enhance protein solubilization. The debris was removed by centrifuging at 2000 g for 10 min and duplicate 0.1-0.4 ml samples of the supernatant were taken for biuret determination of protein while additional 0"5 ml samples were added to 1 ml of 10% trichloroacetic acid. The trichloroacetic acid-protein mixtures were centrifuged after standing overnight and 0'4 ml aliquots of the supernatant were dissolved in scintillation vials with 10 ml of Aquasol (New England Nuclear). The precipitates were washed twice with 5% trichloroacetic acid and twice with 95% ethanol, solubilized by the addition of 0'05 ml water and 0.5 ml of Eastman Tissue Solubilizer and transferred quantitatively into vials with 10 ml of toluene containing 6 g of PPO and 0-5 of POPOP per liter. All samples were counted using Program 8 of a Nuclear Chicago Isocap 300 scintillation counter. The results of the zero min samples were subtracted from the subsequent samples of each experiment to correct for background counts and radioactivity taken up by disrupted cells. All means were compared by the Student's t-test, all variations represent the S.E.M. and in all cases, N = 6.
INTRODUCTION
IT IS well k n o w n that the rates of m a n y metabolic processes are e n h a n c e d during cold acclimation in poikilotherms to compensate for the lower levels of thermal energy available (Fry & Hochachka, 1970). This includes protein synthesis in teleost fishes in vivo (Das, 1967; D e a n & Berlin, 1969; Haschemeyer, 1969b, 1973; Haschemeyer & Persell, 1973; M o r r i s & Smith, 1967; Smith & Morris, 1966) a n d in cell free extracts (Haschemeyer, 1969b). However, in the present study of in vitro protein synthesis in diced livers from temperature acclimated trout, the opposite effect was observed. A possible explanation for this a n o m a l o u s result is provided in this report. MATERIALS AND METHODS Experimental animals The trout, Salma .qairdneri (approx. 100g) were acclimated to 5 and 15°C in 4001 tanks for at least 2 weeks and fed once daily with Clark's rainbow trout pellets. The light regimen was 12 hr light: 12 hr darkness and water was supplied at 1"51/min.
RESULTS
Tissue preparation The fish were stunned, the livers were excised and a sample of the liver was retained for soluble protein determination by the biuret method. The remainder was diced into pieces about 1 mm 3, rinsed 12 times with 5 ml aliquots of rainbow trout saline (Stokes & Fromm, 1964) also containing 5 mM glucose, streptomycin sulphate (100 mg/ml) and penicillin (50 units/ml) and saturated with 95~o 02 : 5~o CO2 (termed the basic medium) and preincubated in 20 ml of this same medium for 1 hr. All incubations contained one liver and were conducted at 10°C using a shaker bath and continuous gassing with the 02 :CO2 mixture.
The up .take of labelled leucine into the trichloroacetic acid soluble fraction was identical for w a r m a n d cold livers (Fig. 1).
Incorporation studies The liver cubes were retrieved on cheesecloth and added to 10 ml of the basic medium containing 0.1 mM leucine, 1.87 #C/ml L-[4,5-H a] leucine (specific activity-29.8 C/mM) and 0.35 #C/ml [carboxyl-C 14] inulin (specific activity2.68 mC/g), both from International Chemical and Nuclear. Samples were withdrawn with a pipet at 0, 20, 40, 60, 90, 120, 150 and 180 min and excess medium was removed by vacuum filtration on glass fiber filters. The samples were rinsed 7 times with 5 ml aliquots of basic medium and then 557
24-5*
× =_
16--
-o
I
I
I
2
P
5
hr
Fig. 1. In vitro uptake at 10°C of L-[4,5-H a] leucine into trichloroaeetic acid soluble material by liver cubes of rainbow trout acclimated to 5 and 15°C. For each point N = 6.
558
TERRAN(?E G . OWEN
i
6-
~!~"1
o.....-~o
I I mo
t I
•
min/mg protein for the warm and cold livers, respectively. DISCUSSION
×
4
I
I
I
2
I
3
hr"
Fig. 2. In vitro uptake at 10°C of [carboxyl-C ~4] inulin by liver cubes of rainbow trout acclimated to 5 and 15°C. For each point N = 6. Vertical bars represent + S.E.
Inulin saturation required about 120min and the warm livers took up 150% more inulin than the cold livers (Fig. 2). The values for inulin uptake were significantly different (P < 0.05) for all sampling times after 40 rain. Comparisons based on the protein determinations were considered to be valid since no significant difference in the protein content per unit wet weight was found between the warm and cold livers. The extracellular space was calculated according to Reiger & Kafatos (1971) and found to be 0'775 + 0-083 and 0"505 + 0'082 p.l/mg protein for warm and cold livers, respectively. These values were significantly different (P < 0.05) and accepted as a true representation of the comparative extracellular space since the H3/ C 14 ratios of the rinses of the labelled tissues were not significantly different from that of the incubation medium. This indicates that inulin was not bound by the tissues (Reiger & Kafatos, 1971). The rate of incorporation of labelled leucine into total protein was 150% greater in the warm livers than the cold livers (Fig. 3). The rates were significantly different (P < 0-01) and were 46 and 31 dis/min per 9 15o
'O
o.
"o
I
2
5
hr
Fig. 3. In vitro incorporation at 10°C of L-[-4,5,H3] leucine into trichloroacetic acid insoluble material by liver cubes of rainbow trout acclimated to 5 and 15°C.The slopes are significantly different (P < 0-01). For each point N = 6.
The greater rate of protein synthesis observed in the warm livers in this investigation seems unusual since other workers have consistently found that the rates of this process are enhanced by cold acclimation (Das, 1967; Dean & Berlin, 1969, Haschemeyer, 1969a,b, 1973; Hascbemeyer & Persell, 1973; Morris & Smith, 1967; Smith & Morris, 1966). However, all of these studies were conducted in vivo with one exception (Haschemeyer, 1969a) in which cell-free extracts were used. Consequently, the different results obtained in the present study are believed to be due to the use ol diced livers, a method that has not been previously exploited for temperature acclimation studies. Although potentially useful in that it is more convenient and offers increased control over experimental conditions compared to in civo studies and still allows examination of the responses of intact tissues, this approach seems to present certain pitfalls when applied to studies of temperature acclimation. This becomes obvious when the anomalous protein synthesis rates are considered. The basis for this reversal of expected results does not appear to reside in the rate of penetration of labelled leucine into the total amino acid pool since these rates are essentially identical (assuming that the specific radioactivities are the same). However, the difference in the rates of protein synthesis coincides exactly to the difference in the extracellular spaces. Also, the precursor pool for protein synthesis appears to be extracellular or in rapid equilibrium with the extracellular pool since the protein synthesis plots are straight lines that pass through the origin (Hider et al., 1969, 1971 ; Kipnis et al., 1961 ; Rosenberg et al., 1963; VanVerooij et al., 1972). Although this conclusion would be supported more conclusively with data from the use of two different isotopic forms of the same amino acid (Alpers & Thier, 1972; Hider et al., 1969, 1971; VanVerooij et al., 1972)it seems valid since there is usually a time lag before a maximum rate of protein synthesis is attained if the precursor pool is intracellular (Alpers & Thier, 1972; Morgan et al., 1971; Reiger & Kafatos, 1971 ; Rosenberg et al., 1963). There may be a direct relationship then, between the volume of the extracellular space and the rate of protein synthesis. The reason for this is not known but if one accepts the hypothesis of Hider et al. (1969) that the external amino acids enter the cellular membrane via "carriers" and are transported directly to the site of protein synthesis without mixing with the intracellular pool, then it seems possible that a reduction of extracellular space could affect the rate of protein synthesis by reducing the number of functional carriers and the amount of amino acids available to the remaining carriers. Since other workers have observed significant, transitory alterations in the extracellular space in teleost tissues as a result of abrupt temperature changes (Heinicke & Houston, 1965; Hickman et al., 1964) it seems particularly possible that such a reduction may have occurred in the cold livers in this study. If so, it is concluded that this paper has illustrated that failure to consider the effects of abrupt temperature changes on the extracellular space in such
Protein synthesis in teleosts temperature acclimation studies could lead to erroneous conclusions. Also, it seems that the in vitro methods described m a y create more problems than they solve despite the theoretical advantages. Obviously, more refinement is required. Acknowledoements--This research was conducted at the University of British Columbia, Vancouver, B.C., Canada and was supported by N. C. R. of Canada grant No. 67-3678 to the author's supervisor of doctoral studies, Dr. P. W. Hochachka of the Zoology Dept., and F. R. B. of Canada grant No. 65-1848.
REFERENCES
ALPERS D. H. & THIER S. O. (1972) Role of the free amino acid pool of the intestine in protein synthesis. Biochim. biophys. Acta 262, 535-545. DAS A. B. (1967) Biochemical changes in tissues of goldfish acclimated to high and low temperatures---II. Synthesis of protein and RNA of subcellular fractions and tissue composition. Comp. Biochem. Physiol. 21,469-485. DEAN J. M. & BERLIN J. D. (1969) Alterations in tiepatocyte function of thermally acclimated rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 29, 307-312. FRY F. E. J. & HOCHACHKA, P. W. (1970) Fish. In Comparative Physiology of Thermoregulation (Edited by WHITTOW G. C.), Vol. I, pp. 79-134. Academic Press, New York. HASCHEMEYERA. E. V. (1969a) Studies on the control of protein synthesis in low temperature acclimation. Comp. Bigchem. Physiol. 28, 535-552. HASCHEMEYERA. E. V. (1969b) Rates of polypeptide chain assembly in liver in vivo: relation to the mechanism of temperature acclimation in Opsanus tau. Proc. natn. Acad. Sci. U.S.A. 62, 128-135. HASCHEMEYERA. E. V. (1973) Kinetic analysis of synthesis and secretion of plasma protein in a marine teleost. J. Biol. Chem. 248, 1643-1649. HASCHE~WR A. E. V. & PERSELL R. (1973) Kinetic studies on amino acid uptake and protein synthesis in liver of temperature acclimated toadfish. Biol. Bull. 145, 472-481.
559
HEINICKE E. A. & HOUSTON A. G. (1965) Effect of thermal acclimation and sublethal heat shock upon ionic regulation in the goldfish, Carassius auratus L. d. Fish Res. Bd Can. 22, 1455-1476. HICKMANC. P. JR., McNABB R. A., NELSON J. S., VANBREEMAN E. D. & CO~FORT D. (1964) Effect of cold acclimation on electrolyte distribution in rainbow trout (Salmo #airdneri). Can. J. Zool. 42, 577-597. HIDER R. C., FERN E. B. & LONDON D. R. (1969) Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats. Biochem. J. 114, 171-178. HIDER R. C., FERN E. B. & LONDON D. R. (1971) Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis. Biochem. J. 121, 817-827. KIPNIS D. M., REISS E. & HELMREICH E. (1961) Functional heterogeneity of the intracellular amino acid pool in mammalian cells. Biochim. biophys. Acta 51,519-524. MORGAN H. E., EARL D. C. N., BROADUS A., WOLPERT E. B., GIGER K. E. & JEFFERSON L. S. (1971) Regulation of protein synthesis in heart muscle--l. Effect of amino acid levels on protein synthesis. J. Biol. Chem. 246, 2152-2162. MORRIS D. & SMITH M. W. (1967) Protein synthesis in the intestine of goldfish acclimatized to different temperatures. Biochem. J. 102, 648-653. RElGER J. C. & KAFATOS F. C. (1971) Microtechnique for determining the specific activity of radioactive intracellular leucine and applications to in vivo studies of protein synthesis. J. Biol. Chem. 246, 6480-6488. ROSENBERGL. E., BERMANM. & SEGAL S. (1963) Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney cortex slices. Biochim. biophys. Acta 71, 664-675. SMITH M. W. & MORRISD. (1966) Temperature acclimatization and protein synthesis in the goldfish mucosa. Experientia 22, 678-679. STORES R. M. & FROMM P. O. (1964) Glucose absorption and metabolism by the gut of rainbow trout. Comp. Bigchem. Physiol. 13, 53-69. VANVEROOIJW. J., POORT C., KRAMER M. F. & JANSEN M. T. (1972) Relationship between extracellular amino acids and protein synthesis in vitro in the rat pancreas. Eur. J. Biochem. 30, 427-433. Key Word Index--Extracellular space; protein synthesis; temperature acclimation.