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Temperature adaptation of lysosomal enzymes in fishes
Corao. Biochera. Physiol. Vol. 61B, pp. 115-118 © Pergamon Press Ltd., 1978. Printed in Great Britain
0305-0491/78/0815-01151502.00/0
T E M P E R A T U R E ADAPTATION OF LYSOSOMAL ENZYMES IN FISHES BIRGIT HELENE DANNEVIG* and TROND BERG~ *Institute of Zoophysiology and ?Institute for Nutrition Research, University of Oslo, Blindern, Oslo 3, Norway
(Received 27 October 1977) Abstract--l. The apparent Michaelis constants (K,.) of the substrates for two lysosomal enzymes (/3-acetylglucosaminidase and cathepsin D) were measured in lysosome-rich liver fractions from hagtish, char and rat. The Ks-values were considerably lower for the fish enzymes than for the homologous rat enzymes. 2. Activation energies (Eo) of/]-acetylglucosaminidase measured in the fish preparations were slightly lower than the F_~ of the rat enzyme. Eo of cathepsin D was higher for the char enzyme than for the rat cathepsin D. 3. The specific activities of the two enzymes (measured at 37°C) were more than 4 times higher in liver homogenates from hagfish and char than in rat liver homogenates.
INTRODUCTION L y s o s o m e s in fish tissues must function in thermal conditions different than those in mammalian tissues. P r o c e s s e s in the lysosomal system which may be influenced by temperature are the m e c h a n i s m s which transport the substrates into the l y s o s o m e s (endocytosis or autophagy) and the degradation of the substrates by the lysosomal acid hydrolases. Fishes living in northern E u r o p e are e x p o s e d to low temperatures in the winter or through the whole year. It was of interest to find out if lysosomal e n z y m e s f r o m cold-adapted fishes exhibited kinetic or t h e r m o d y n a m i c properties which could c o m p e n s a t e for the low thermic energy available for the chemical reactions. In this study we have m e a s u r e d the substrate affinities of two lysosomal e n z y m e s , expressed as the apparent K,, of their specific substrates, and the activation energies (Ea) of the same lysosomal e n z y m e s f r o m livers of hagfish (Myxine glutinosa L.), char (Salmo alphinus L.) and rat. In addition, the specific activities of the e n z y m e s were measured. High enzyme concentration, as m e a s u r e d by the specific activities, may also make the catalytic potential higher. The hagfish is normally living at 5-10°C (Adam & Strahan, 1963), but the char is exposed to seasonal temperature variations. The chars used were captured in the winter and held in fresh water at 5°C for several w e e k s b e f o r e use. The rat liver e n z y m e s represent homologous warm-adapted e n z y m e s , and were included in the experiments for c o m p a r a t i v e purposes. The lysosomal e n z y m e s e x a m i n e d w e r e flacetylglucosaminidase and cathepsin D. A crude, lysosome-rich fraction prepared by differential centrifugation was used as the e n z y m e source for measuring the K= and F_~.
MATERIALS AND METHODS
Animals Hagtish (Myxine glutinosa L.) were obtained from the Biological Station, Dr6bak, Norway. The animals ranged from 25 to 35 cm in length. Both sexes were used. They were kept in fresh, aerated sea water at 5°C. No food was given. Male and female chars (Salmo alpinus L ) 5 yr of age, weighing about 200-300 g were obtained from a station for char investigation at Voss, Norway. The animals were kept in running, aerated tap water at 5"C and given food ad libitum (Ewos salmon food no. 4, Str6msn~is, Sweden). Male Wistar rats, about 200 g, were from Mi~llegaard Hansens Avlslahoratorier A/S, Ejby, Denmark. The animals were given ordinary lab chow and water ad
libitum. Homogenization of livers The fishes were not anaesthetized before dissection. The rat liver was taken out under light ether anaesthesia. The livers were homogenized in nine volumes of icecold, isotonic sucrose solution with a Potter-Elvehjem homogenizer with a teflon pestle. The tissue was kept in an ice-slurry during the homogenizing procedure which consisted of two down-up strokes of the pestle rotating at about 500 rev/min. The final homogenate was filtered through a double layer of gas to remove connective tissue and tissue debris.
Preparation of lysosome-rich liver fractions by differential centrifugation Fractionation of the homogenate (5 g wet tissue/100 ml isotonic sucrose) was carried out in a Sorvall RC-2B centrifuge with angle rotor SM-24 according to De Duve etal. (1955). The temperature was 0-2°C in the centrifuge. The pellets were washed once in isotonic sucrose at each step in the centrifugation procedure. The resulting fractions were the nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and the soluble fraction (S). In preliminary experiments we found that the lysosomal enzyme ~-acetylglucosaminidase showed a higher relative specific activity in the M fractions than in I15
116
BIRGIT HELENE DANNEVIG and TROND BERG
the L fractions from livers of hagtish and char. /3Acetylglucosaminidase showed a typical lysosomai distribution in fractions prepared in an identical fashion from rat liver. Because of the high enrichment of the enzyme in the mitochondrial fraction from the fishes, we decided to use the mitochondrial and the light mitochondrial fractions together as a "lysosome-rich" fraction for all animals in the following experiments.
Biochemical determinations Enzyme substrates were from Sigma Chemical Co., St. Louis, U.S.A. /3-Acetylglucosaminidase (EC. 3.2.1.30) was measured according to Barrett (1972). The assay mixture contained, in a total volume of 0.75 ml, 5 mM p-nitrophenyI-Nacetyl-~,D-glucosaminide, 0. ! M sodium citrate-citric acid buffer with 0.1 M sodium chloride, pH 4.3, and 0.25 ml tissue homogenate or lysosome-rich liver preparation. It was the maximal enzyme activity which was assayed when the substrate concentration was 5 mM in the incubation mixture. Cathepsin D (EC. 3.4.4.23) was determined by the method of Anson (1937) as modified by Barrett (1972). The assay mixture contained, in a total volume of 0.5 ml, 2% (w/v) bovine hemoglobin (when maximal enzyme activity was measured), 0.25M formic acid-sodium formate buffer, pH 3.2, and 0.25 ml tissue preparation. For all enzyme measurements it was checked that the enzyme activities were linear with time and enzyme concentration under the assay conditions used. The activities were measured at the pH-optima of the enzymes, which were the same for the homologous enzymes from the experimental animals. In order to measure "total" lysosomal enzyme activity, the organelles were disrupted by diluting the homogenates with distilled water. Protein was measured according to Lowry et al. (1951). The apparent Michaelis constant (Kin) was determined from double-reciprocal plots (Lineweaver-Burk plots). The enzyme activities were measured at five different substrate concentrations to estimate K= at each tempera.ture tested. Lysosome-rich liver fractions were used as tissue preparations in these cases. Both enzymes exhibited classical hyperbolic saturation curves with respect to substrate concentration. The activation energies (E=) of /3-acetylglucosaminidase and cathepsin D were determined from the slopes of Arrhenius plots (log Vmax vs l/absolute temperature). Vmaxwas measured in lysosome-rich fractions at five different temperatures in the range of 545°C, for each determination of E=. The Arrhenius plots were linear over the aforementioned temperature range. In order to calculate the Km and E= values, the regression lines were determined for each experiment. The correlation coefficients of the regression lines were all higher than 0.94.
Substrate aBinities of/3-acetylglucosaminidase and cathepsin D in livers from fish and rat The Km of p-nitrophenylglucosaminide for /3acetylglucosaminidase was measured at three different temperatures for all animals as shown in Fig. 1. The Km of this substrate for the hagfish enzyme was 0.19, 0.21 and 0.29 mM at the respective temperatures 4, 13 and 26°C. K s at 26°C was significantly higher than Km measured at 4 and 13°C. The corresponding Km values for /3acetylglucosaminidase prepared from char liver were about 0.15 mM at all experimental temperatures (4, 13 and 26°C). Determination of Km of the same substrate f o r the enzyme prepared from rat liver fractions was carried out at 4, 13 and 37°C. The values for the homologous rat enzyme were 0.67, 0.73 and 0.75 raM, respectively. T h e r e were no significant variations in the Km values at the different temperatures for the rat enzyme. The results show that the affinity of /3-acetylglucosaminidase for the specific substrate used was 3-4 times higher for the fish enzymes compared to the homologous rat enzyme at all experimental temperatures (Fig. 1). In the case of cathepsin D, the Km of the substrate bovine hemoglobin was determined in lysosome-rich liver fractions from char and rat only. The measurements were carried out once at the temperatures shown in Fig. 2. The Km of bovine hemoglobin for the char enzyme was 0.34 x 10-5, 0.28 × 10-5 and 0.49 x 10-5 M at the temperatures 4, 13 and 37°C, respectively. The Km values for the rat cathepsin D were 0.83 x 10-5 M at 13°C and 0.75 x 10-~M at 37°C. Thus, also in this case was substrate affinity higher for the fish enzyme than that from the rat at the experimental temperatures used.
Activation energies (Ea) relationship The activation energies of /3-acetylglucosaminidase and cathepsin D measured in lysosome-rich liver fractions from haglish, char and rat are given in Table 2. The activation energy of/3-acetylglucosaminidase from all three species were very similar. The values ranged from about l l,600cal/mole for the fish enzymes to about
ID
Rat (n=3] }
RESULTS
Activities of lysosomal enzymes in liver homogenates from fish and rat Table 1 shows the specific activities (nmoles/min/mg protein) for /3-acetylglucosaminidase and cathepsin D measured in liver-homogenates from hagiish, char and rat. It is apparent that the activities of /3-acetylglucosaminidase were 4--6 times higher in livers from the fishes than in rat liver. In the case of cathepsin D, the enzyme activities were 5-8 times higher in fish livers than in rat liver. Considerable variations in the specific activities in livers from different fishes were observed.
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~
Hogfish(n=4) Char(n=3)
~
I
i
I
J
i
I
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5
10
15
20
25
30
35
40
Temperature,
°C
Fig. 1. K,, of p-nitrophenylglucosaminid¢ for /3-acetylgiucosaminidase in lysosome-rich liver fractions from hagfish, char and rat measured at different temperatures. The values given represent means -+S.D. for the number (n) of experimental animals given in the figure.
Temperature adaptation of iysosomal enzymes in fishes
117
Table 1. Specific activities of ~-acetylglucosaminidase and cathepsin D in livers from hagfish, char and rat
Enzyme ~Acetylglucosaminidase Cathepsin D
Hagtish
Char
(5) 87.3-+14.3. (5) 131.8-+58.8 (4) 104.5-+21.5 (3) 72.5-+9.5
Rat (3) 23.1-+2.7 (3) 13.8-+0.4
Total enzyme activities were measured in liver homogenates diluted with distilled water, and are expressed as nmoles/min per mg protein. The values given represent means -+S.D. for the number of experimental animals given in parenthesis. The enzyme activities were measured at 37"C. Km values ( h i g h affinities), then the enzymes would be saturated with substrate at very low substrate concentrations. In this situation the =~ Rat" regulation of enzyme activity would be restricted. Instead, it has been suggested by Hochachka & Somero (1973) that the Km values are adapted in s_ such a way that the regulatory function of the enzymes is optimal, which may be particularly important for enzymes concerned with the energy production of the cell. Our data on the Km of the substrates for both /3-acetylglucosaminidase and cathepsin D indicate that the enzyme-substrate affinities are considerably higher for the fish enI I I I I I I I O 5 I0 15 20 25 30 55 40 zymes than for the rat enzymes. The K~, values Temperature, *C showed little or no variation with experimental Fig. 2. Km of bovine hemoglobin for cathepsin D in temperature (Figs. I and 2). These high affinities may lysosome-rich liver fractions at different experimental partially compensate for the low temperature the temperatures. The measurements were carried out once fishes are exposed to, if the substrate concentration at each temperature. i n the lysosomes is lower than that which gives the Vm~ for the enzyme reaction. In an earlier study (Dannevig & Berg, 1978) we have found that the Table 2. Activation energy (E=) values of ~-acetyl- uptake of substrate into the lysosomes was much glucosaminidase and cathepsin D measured in iysosome- slower in char kidney lysosomes at 7"C than in rat rich liver fractions from hagfish, car and rat liver lysosomes at 37°C. This indicates that the substrate concentration is lower in char lysosomes Eo (callmole) than in rat lysosomes. Animal ~-Acetylglucosaminidase CathepsinD • H a t e r et al. (1975) have observed a positive correlation between the preferred temperatures of Hagfish (4) 11,700-+200 Char (4) 11,600-+600 (1) 12,700 several ectothermic vertebrates and the substrate Rat (4) 13,200-+500 (1) 7700 affinities of their trypsins. Both the intracellular lysosomal enzymes and the extracellular trypsins The values are means -+S.D. for the number of animals are digestive enzymes. Their function is to degrade given in parenthesis. the substrates available at any time, and their role in the regulation of the energy metabolism of the 13,200cal/mole for the rat enzyme. There was a cell may be rather small. In view of this, the high significant difference between the Eo-values of the enzyme-substrate affinities for the fish enzymes fish and rat enzymes, but the real difference was may be an important factor in temperature adaprather small. tation for lysosomal enzymes in fishes. As opposed to /3-acetylglucosaminidase, the The activation energies of t3-acetylactivation energy of reactions catalyzed by glucosaminidase from the fishes were significantly cathepsin D from char and rat liver showed lower than for the homologous rat enzyme, but considerable differences, and it is remarkable that the difference was rather small (Table 1). In the the Eo value of cathepsin D from the char case of cathepsin D, the Eo value was 1.5 times (12,500 cal/mole) was 1.5 times higher than the Eo higher for the char enzyme than for t h e rat envalue of the rat enzyme (7500 cal/mole) (Table 2). zyme. Our results may suggest that the energy barrier (represented by the Ea value) is not a limiting step in the reaction procedure for these DISCUSSION lysosomal enzymes. Hochachka & Somero (1973) have discussed The Ea value, which is an enthalpy derived enzyme-substrate affinity, activation energy and parameter, has often been used as a quantitative enzyme concentration as possible compensatory measure of the ability of the enzymes to reduce mechanisms for biochemical temperature adap- the energy barrier of a specific reaction. A positive tation. Their data indicate that, in the case of some correlation between the Ea values and adaptation enzymes, the Km of their substrates cannot be temperature of the animals has been observed in positively correlated to the animal's adaptation many cases (Hochachka & Somero, 1971; Low et temperatures. If cold adaptation should imply low al., 1973). However, it is now well accepted that it 1.0
118
BIROIT HELENE DANNEVIG and TROND BERG
is the free energy of activation which determines the efficiency of the enzymes as catalysts (Low et al., 1973). Since the free energy of activation depends both on the activation enthalpy and the activation entropy, it may be concluded that the Ea value can only give an indication of the thermodynamical properties of homologous enzymes (Hochachka & Somero, 1971). The specific activities of the lysosomal enzymes measured were remarkably higher in fish liver than in rat liver. High enzyme concentrations may imply that more substrate molecules could undergo their specific reactions at a given temperature. It has been discussed by Hochachka & Somero (1973) that high enzyme concentration is not a "good" solution of the problem of evolutionary temperature adaptation, since the solvent capacity of the cell may be a limiting factor (Atkinson, 1969). Not much attention has been paid to enzyme concentrations (activities) in animals adapted to different thermal environments. Boernke (1977) has measured the arginase maximum velocities in several ectotherms and endotherms. He found that the enzyme activities were higher in livers from amphibians than in mammalian livers, and suggests that this must be important in the adaptation process to the ectothermic way of life. In addition, Wilson et al. (1974) have found about 70% higher activities of cytochrome oxidase in a rockfish species (Sebastes miniatus) which lives in cool water below the thermocline, than in the closely related species S. auriculatus which lives in shallow water and is then exposed to temperatures over a wider range. In conclusion, the fish lysosomal enzymes seem to be well suited to function in the thermal environments of these experimental animals, because of the high enzyme-substrate affinities and the high enzyme activities of the two lysosomal enzymes examined. The lysosomal enzymes get in contact with their specific substrates by the process of endocytosis (De Duve, 1969). As mentioned previously, the uptake of substrate into the char lysosomes was considerably lower than in the rat lysosomes. Thus, the high enzyme--substrate affinity of the char lysosomal enzymes may be of great advantage in ensuring a sufficient turnover of substrate.
REFERENCES ADAM H. • STRAHANR. (1963) Notes on the habitat, aquarium maintenance, and experimental use of hagfish. In The Biology of Myxine (Edited by BRODAL A. & F.~NGER.), pp. 33--41. Universitetsforlaget, Oslo. ANSON M. L. (1937) The estimation of cathepsin with hemoglobin and the partial purification of cathepsin. J. gen. Physiol. 20, 565-574. AaYONSOND. E. (1969) Limitation of metabolite concentrations and the conservation of solvent capacity in the living cell. Curr. Topics Cell. Regul. 1, 29--43. B.~dtm~r A. J. (1972) Lysosomal enzymes. In Lysosomes--A Laboratory Handbook (Edited by DINGLEJ. T.), pp. 46--135. North-Holland, Amsterdam. BOERNKE W. E. (1977) A comparison of arginase maximum velocities from several poikilotherms and homeotherms. Comp. Biochem. Physiol. 5611, 113--116. DANNEVm B. H. & BERGT. (1978) Uptake and proteolysis of denatured human serum albumin by kidneys in chars (Salmo alpinus L.). Comp. Biochem. Physiol. 59A, 299-303. DE DUVE C. (1969) The lysosome in retrospect. In Lysosomes in Biology and Pathology (Edited by DINGLE J. T. & FELL H. B.), Vo[. 1, pp. 3--40. NorthHolland, Amsterdam. DE DUVE C., PRESSMANB. C., GIANE'rrOR., WATTIAUX R. & APPELMANSF. (1955) Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissues. Biochem. J. 60, 604-617. HOCHACHKAP. W. & SOMEROG. N. (1971) Biochemical adaptation to the environment. In Fish Physhglogy, VI (Edited by HOARW. S. & RANDALLn. J.), pp. 99-156. Academic Press, New York. HOCHACHKAP. W. & SOMEROG. N. (1973) Strategies of Biochemical Adaptation, p. 358. W. B. Saunders, Philadelphia. HOFER R., LADURNERH., GATrR1NGERA. & WlESERW. (1975) Relationship between the temperature preferenda of fish, amphibians and reptiles, and the substrate affinities of their trypsins. J. comp. Physiol. 99, 345-355. Low P. S., BADAJ. L. & SOMEROG. N. (1973) Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. Proc. natn. Acad. Sci. U.S.A. 70(2), 431)--432. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. WILSON F. R., SOMEROG. N. & PROSSER C. L. (1974) Temperature-metabolism relations of two species of Sebastes from different thermal environments. Comp. Biochem. Physiol. 47B, 485-491.
0305-0491/78/0815-01151502.00/0
T E M P E R A T U R E ADAPTATION OF LYSOSOMAL ENZYMES IN FISHES BIRGIT HELENE DANNEVIG* and TROND BERG~ *Institute of Zoophysiology and ?Institute for Nutrition Research, University of Oslo, Blindern, Oslo 3, Norway
(Received 27 October 1977) Abstract--l. The apparent Michaelis constants (K,.) of the substrates for two lysosomal enzymes (/3-acetylglucosaminidase and cathepsin D) were measured in lysosome-rich liver fractions from hagtish, char and rat. The Ks-values were considerably lower for the fish enzymes than for the homologous rat enzymes. 2. Activation energies (Eo) of/]-acetylglucosaminidase measured in the fish preparations were slightly lower than the F_~ of the rat enzyme. Eo of cathepsin D was higher for the char enzyme than for the rat cathepsin D. 3. The specific activities of the two enzymes (measured at 37°C) were more than 4 times higher in liver homogenates from hagfish and char than in rat liver homogenates.
INTRODUCTION L y s o s o m e s in fish tissues must function in thermal conditions different than those in mammalian tissues. P r o c e s s e s in the lysosomal system which may be influenced by temperature are the m e c h a n i s m s which transport the substrates into the l y s o s o m e s (endocytosis or autophagy) and the degradation of the substrates by the lysosomal acid hydrolases. Fishes living in northern E u r o p e are e x p o s e d to low temperatures in the winter or through the whole year. It was of interest to find out if lysosomal e n z y m e s f r o m cold-adapted fishes exhibited kinetic or t h e r m o d y n a m i c properties which could c o m p e n s a t e for the low thermic energy available for the chemical reactions. In this study we have m e a s u r e d the substrate affinities of two lysosomal e n z y m e s , expressed as the apparent K,, of their specific substrates, and the activation energies (Ea) of the same lysosomal e n z y m e s f r o m livers of hagfish (Myxine glutinosa L.), char (Salmo alphinus L.) and rat. In addition, the specific activities of the e n z y m e s were measured. High enzyme concentration, as m e a s u r e d by the specific activities, may also make the catalytic potential higher. The hagfish is normally living at 5-10°C (Adam & Strahan, 1963), but the char is exposed to seasonal temperature variations. The chars used were captured in the winter and held in fresh water at 5°C for several w e e k s b e f o r e use. The rat liver e n z y m e s represent homologous warm-adapted e n z y m e s , and were included in the experiments for c o m p a r a t i v e purposes. The lysosomal e n z y m e s e x a m i n e d w e r e flacetylglucosaminidase and cathepsin D. A crude, lysosome-rich fraction prepared by differential centrifugation was used as the e n z y m e source for measuring the K= and F_~.
MATERIALS AND METHODS
Animals Hagtish (Myxine glutinosa L.) were obtained from the Biological Station, Dr6bak, Norway. The animals ranged from 25 to 35 cm in length. Both sexes were used. They were kept in fresh, aerated sea water at 5°C. No food was given. Male and female chars (Salmo alpinus L ) 5 yr of age, weighing about 200-300 g were obtained from a station for char investigation at Voss, Norway. The animals were kept in running, aerated tap water at 5"C and given food ad libitum (Ewos salmon food no. 4, Str6msn~is, Sweden). Male Wistar rats, about 200 g, were from Mi~llegaard Hansens Avlslahoratorier A/S, Ejby, Denmark. The animals were given ordinary lab chow and water ad
libitum. Homogenization of livers The fishes were not anaesthetized before dissection. The rat liver was taken out under light ether anaesthesia. The livers were homogenized in nine volumes of icecold, isotonic sucrose solution with a Potter-Elvehjem homogenizer with a teflon pestle. The tissue was kept in an ice-slurry during the homogenizing procedure which consisted of two down-up strokes of the pestle rotating at about 500 rev/min. The final homogenate was filtered through a double layer of gas to remove connective tissue and tissue debris.
Preparation of lysosome-rich liver fractions by differential centrifugation Fractionation of the homogenate (5 g wet tissue/100 ml isotonic sucrose) was carried out in a Sorvall RC-2B centrifuge with angle rotor SM-24 according to De Duve etal. (1955). The temperature was 0-2°C in the centrifuge. The pellets were washed once in isotonic sucrose at each step in the centrifugation procedure. The resulting fractions were the nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and the soluble fraction (S). In preliminary experiments we found that the lysosomal enzyme ~-acetylglucosaminidase showed a higher relative specific activity in the M fractions than in I15
116
BIRGIT HELENE DANNEVIG and TROND BERG
the L fractions from livers of hagtish and char. /3Acetylglucosaminidase showed a typical lysosomai distribution in fractions prepared in an identical fashion from rat liver. Because of the high enrichment of the enzyme in the mitochondrial fraction from the fishes, we decided to use the mitochondrial and the light mitochondrial fractions together as a "lysosome-rich" fraction for all animals in the following experiments.
Biochemical determinations Enzyme substrates were from Sigma Chemical Co., St. Louis, U.S.A. /3-Acetylglucosaminidase (EC. 3.2.1.30) was measured according to Barrett (1972). The assay mixture contained, in a total volume of 0.75 ml, 5 mM p-nitrophenyI-Nacetyl-~,D-glucosaminide, 0. ! M sodium citrate-citric acid buffer with 0.1 M sodium chloride, pH 4.3, and 0.25 ml tissue homogenate or lysosome-rich liver preparation. It was the maximal enzyme activity which was assayed when the substrate concentration was 5 mM in the incubation mixture. Cathepsin D (EC. 3.4.4.23) was determined by the method of Anson (1937) as modified by Barrett (1972). The assay mixture contained, in a total volume of 0.5 ml, 2% (w/v) bovine hemoglobin (when maximal enzyme activity was measured), 0.25M formic acid-sodium formate buffer, pH 3.2, and 0.25 ml tissue preparation. For all enzyme measurements it was checked that the enzyme activities were linear with time and enzyme concentration under the assay conditions used. The activities were measured at the pH-optima of the enzymes, which were the same for the homologous enzymes from the experimental animals. In order to measure "total" lysosomal enzyme activity, the organelles were disrupted by diluting the homogenates with distilled water. Protein was measured according to Lowry et al. (1951). The apparent Michaelis constant (Kin) was determined from double-reciprocal plots (Lineweaver-Burk plots). The enzyme activities were measured at five different substrate concentrations to estimate K= at each tempera.ture tested. Lysosome-rich liver fractions were used as tissue preparations in these cases. Both enzymes exhibited classical hyperbolic saturation curves with respect to substrate concentration. The activation energies (E=) of /3-acetylglucosaminidase and cathepsin D were determined from the slopes of Arrhenius plots (log Vmax vs l/absolute temperature). Vmaxwas measured in lysosome-rich fractions at five different temperatures in the range of 545°C, for each determination of E=. The Arrhenius plots were linear over the aforementioned temperature range. In order to calculate the Km and E= values, the regression lines were determined for each experiment. The correlation coefficients of the regression lines were all higher than 0.94.
Substrate aBinities of/3-acetylglucosaminidase and cathepsin D in livers from fish and rat The Km of p-nitrophenylglucosaminide for /3acetylglucosaminidase was measured at three different temperatures for all animals as shown in Fig. 1. The Km of this substrate for the hagfish enzyme was 0.19, 0.21 and 0.29 mM at the respective temperatures 4, 13 and 26°C. K s at 26°C was significantly higher than Km measured at 4 and 13°C. The corresponding Km values for /3acetylglucosaminidase prepared from char liver were about 0.15 mM at all experimental temperatures (4, 13 and 26°C). Determination of Km of the same substrate f o r the enzyme prepared from rat liver fractions was carried out at 4, 13 and 37°C. The values for the homologous rat enzyme were 0.67, 0.73 and 0.75 raM, respectively. T h e r e were no significant variations in the Km values at the different temperatures for the rat enzyme. The results show that the affinity of /3-acetylglucosaminidase for the specific substrate used was 3-4 times higher for the fish enzymes compared to the homologous rat enzyme at all experimental temperatures (Fig. 1). In the case of cathepsin D, the Km of the substrate bovine hemoglobin was determined in lysosome-rich liver fractions from char and rat only. The measurements were carried out once at the temperatures shown in Fig. 2. The Km of bovine hemoglobin for the char enzyme was 0.34 x 10-5, 0.28 × 10-5 and 0.49 x 10-5 M at the temperatures 4, 13 and 37°C, respectively. The Km values for the rat cathepsin D were 0.83 x 10-5 M at 13°C and 0.75 x 10-~M at 37°C. Thus, also in this case was substrate affinity higher for the fish enzyme than that from the rat at the experimental temperatures used.
Activation energies (Ea) relationship The activation energies of /3-acetylglucosaminidase and cathepsin D measured in lysosome-rich liver fractions from haglish, char and rat are given in Table 2. The activation energy of/3-acetylglucosaminidase from all three species were very similar. The values ranged from about l l,600cal/mole for the fish enzymes to about
ID
Rat (n=3] }
RESULTS
Activities of lysosomal enzymes in liver homogenates from fish and rat Table 1 shows the specific activities (nmoles/min/mg protein) for /3-acetylglucosaminidase and cathepsin D measured in liver-homogenates from hagiish, char and rat. It is apparent that the activities of /3-acetylglucosaminidase were 4--6 times higher in livers from the fishes than in rat liver. In the case of cathepsin D, the enzyme activities were 5-8 times higher in fish livers than in rat liver. Considerable variations in the specific activities in livers from different fishes were observed.
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~
Hogfish(n=4) Char(n=3)
~
I
i
I
J
i
I
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5
10
15
20
25
30
35
40
Temperature,
°C
Fig. 1. K,, of p-nitrophenylglucosaminid¢ for /3-acetylgiucosaminidase in lysosome-rich liver fractions from hagfish, char and rat measured at different temperatures. The values given represent means -+S.D. for the number (n) of experimental animals given in the figure.
Temperature adaptation of iysosomal enzymes in fishes
117
Table 1. Specific activities of ~-acetylglucosaminidase and cathepsin D in livers from hagfish, char and rat
Enzyme ~Acetylglucosaminidase Cathepsin D
Hagtish
Char
(5) 87.3-+14.3. (5) 131.8-+58.8 (4) 104.5-+21.5 (3) 72.5-+9.5
Rat (3) 23.1-+2.7 (3) 13.8-+0.4
Total enzyme activities were measured in liver homogenates diluted with distilled water, and are expressed as nmoles/min per mg protein. The values given represent means -+S.D. for the number of experimental animals given in parenthesis. The enzyme activities were measured at 37"C. Km values ( h i g h affinities), then the enzymes would be saturated with substrate at very low substrate concentrations. In this situation the =~ Rat" regulation of enzyme activity would be restricted. Instead, it has been suggested by Hochachka & Somero (1973) that the Km values are adapted in s_ such a way that the regulatory function of the enzymes is optimal, which may be particularly important for enzymes concerned with the energy production of the cell. Our data on the Km of the substrates for both /3-acetylglucosaminidase and cathepsin D indicate that the enzyme-substrate affinities are considerably higher for the fish enI I I I I I I I O 5 I0 15 20 25 30 55 40 zymes than for the rat enzymes. The K~, values Temperature, *C showed little or no variation with experimental Fig. 2. Km of bovine hemoglobin for cathepsin D in temperature (Figs. I and 2). These high affinities may lysosome-rich liver fractions at different experimental partially compensate for the low temperature the temperatures. The measurements were carried out once fishes are exposed to, if the substrate concentration at each temperature. i n the lysosomes is lower than that which gives the Vm~ for the enzyme reaction. In an earlier study (Dannevig & Berg, 1978) we have found that the Table 2. Activation energy (E=) values of ~-acetyl- uptake of substrate into the lysosomes was much glucosaminidase and cathepsin D measured in iysosome- slower in char kidney lysosomes at 7"C than in rat rich liver fractions from hagfish, car and rat liver lysosomes at 37°C. This indicates that the substrate concentration is lower in char lysosomes Eo (callmole) than in rat lysosomes. Animal ~-Acetylglucosaminidase CathepsinD • H a t e r et al. (1975) have observed a positive correlation between the preferred temperatures of Hagfish (4) 11,700-+200 Char (4) 11,600-+600 (1) 12,700 several ectothermic vertebrates and the substrate Rat (4) 13,200-+500 (1) 7700 affinities of their trypsins. Both the intracellular lysosomal enzymes and the extracellular trypsins The values are means -+S.D. for the number of animals are digestive enzymes. Their function is to degrade given in parenthesis. the substrates available at any time, and their role in the regulation of the energy metabolism of the 13,200cal/mole for the rat enzyme. There was a cell may be rather small. In view of this, the high significant difference between the Eo-values of the enzyme-substrate affinities for the fish enzymes fish and rat enzymes, but the real difference was may be an important factor in temperature adaprather small. tation for lysosomal enzymes in fishes. As opposed to /3-acetylglucosaminidase, the The activation energies of t3-acetylactivation energy of reactions catalyzed by glucosaminidase from the fishes were significantly cathepsin D from char and rat liver showed lower than for the homologous rat enzyme, but considerable differences, and it is remarkable that the difference was rather small (Table 1). In the the Eo value of cathepsin D from the char case of cathepsin D, the Eo value was 1.5 times (12,500 cal/mole) was 1.5 times higher than the Eo higher for the char enzyme than for t h e rat envalue of the rat enzyme (7500 cal/mole) (Table 2). zyme. Our results may suggest that the energy barrier (represented by the Ea value) is not a limiting step in the reaction procedure for these DISCUSSION lysosomal enzymes. Hochachka & Somero (1973) have discussed The Ea value, which is an enthalpy derived enzyme-substrate affinity, activation energy and parameter, has often been used as a quantitative enzyme concentration as possible compensatory measure of the ability of the enzymes to reduce mechanisms for biochemical temperature adap- the energy barrier of a specific reaction. A positive tation. Their data indicate that, in the case of some correlation between the Ea values and adaptation enzymes, the Km of their substrates cannot be temperature of the animals has been observed in positively correlated to the animal's adaptation many cases (Hochachka & Somero, 1971; Low et temperatures. If cold adaptation should imply low al., 1973). However, it is now well accepted that it 1.0
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BIROIT HELENE DANNEVIG and TROND BERG
is the free energy of activation which determines the efficiency of the enzymes as catalysts (Low et al., 1973). Since the free energy of activation depends both on the activation enthalpy and the activation entropy, it may be concluded that the Ea value can only give an indication of the thermodynamical properties of homologous enzymes (Hochachka & Somero, 1971). The specific activities of the lysosomal enzymes measured were remarkably higher in fish liver than in rat liver. High enzyme concentrations may imply that more substrate molecules could undergo their specific reactions at a given temperature. It has been discussed by Hochachka & Somero (1973) that high enzyme concentration is not a "good" solution of the problem of evolutionary temperature adaptation, since the solvent capacity of the cell may be a limiting factor (Atkinson, 1969). Not much attention has been paid to enzyme concentrations (activities) in animals adapted to different thermal environments. Boernke (1977) has measured the arginase maximum velocities in several ectotherms and endotherms. He found that the enzyme activities were higher in livers from amphibians than in mammalian livers, and suggests that this must be important in the adaptation process to the ectothermic way of life. In addition, Wilson et al. (1974) have found about 70% higher activities of cytochrome oxidase in a rockfish species (Sebastes miniatus) which lives in cool water below the thermocline, than in the closely related species S. auriculatus which lives in shallow water and is then exposed to temperatures over a wider range. In conclusion, the fish lysosomal enzymes seem to be well suited to function in the thermal environments of these experimental animals, because of the high enzyme-substrate affinities and the high enzyme activities of the two lysosomal enzymes examined. The lysosomal enzymes get in contact with their specific substrates by the process of endocytosis (De Duve, 1969). As mentioned previously, the uptake of substrate into the char lysosomes was considerably lower than in the rat lysosomes. Thus, the high enzyme--substrate affinity of the char lysosomal enzymes may be of great advantage in ensuring a sufficient turnover of substrate.
REFERENCES ADAM H. • STRAHANR. (1963) Notes on the habitat, aquarium maintenance, and experimental use of hagfish. In The Biology of Myxine (Edited by BRODAL A. & F.~NGER.), pp. 33--41. Universitetsforlaget, Oslo. ANSON M. L. (1937) The estimation of cathepsin with hemoglobin and the partial purification of cathepsin. J. gen. Physiol. 20, 565-574. AaYONSOND. E. (1969) Limitation of metabolite concentrations and the conservation of solvent capacity in the living cell. Curr. Topics Cell. Regul. 1, 29--43. B.~dtm~r A. J. (1972) Lysosomal enzymes. In Lysosomes--A Laboratory Handbook (Edited by DINGLEJ. T.), pp. 46--135. North-Holland, Amsterdam. BOERNKE W. E. (1977) A comparison of arginase maximum velocities from several poikilotherms and homeotherms. Comp. Biochem. Physiol. 5611, 113--116. DANNEVm B. H. & BERGT. (1978) Uptake and proteolysis of denatured human serum albumin by kidneys in chars (Salmo alpinus L.). Comp. Biochem. Physiol. 59A, 299-303. DE DUVE C. (1969) The lysosome in retrospect. In Lysosomes in Biology and Pathology (Edited by DINGLE J. T. & FELL H. B.), Vo[. 1, pp. 3--40. NorthHolland, Amsterdam. DE DUVE C., PRESSMANB. C., GIANE'rrOR., WATTIAUX R. & APPELMANSF. (1955) Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissues. Biochem. J. 60, 604-617. HOCHACHKAP. W. & SOMEROG. N. (1971) Biochemical adaptation to the environment. In Fish Physhglogy, VI (Edited by HOARW. S. & RANDALLn. J.), pp. 99-156. Academic Press, New York. HOCHACHKAP. W. & SOMEROG. N. (1973) Strategies of Biochemical Adaptation, p. 358. W. B. Saunders, Philadelphia. HOFER R., LADURNERH., GATrR1NGERA. & WlESERW. (1975) Relationship between the temperature preferenda of fish, amphibians and reptiles, and the substrate affinities of their trypsins. J. comp. Physiol. 99, 345-355. Low P. S., BADAJ. L. & SOMEROG. N. (1973) Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. Proc. natn. Acad. Sci. U.S.A. 70(2), 431)--432. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. WILSON F. R., SOMEROG. N. & PROSSER C. L. (1974) Temperature-metabolism relations of two species of Sebastes from different thermal environments. Comp. Biochem. Physiol. 47B, 485-491.