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Effect of salinity on the activities of digestive proteases from the tilapia fish, Oreochromis niloticus in different culture environments
Camp. Biochem. Physiol. Vol. 93A, No. 2, pp. 439443, Printed in Great Britain
1989
0300-9629/89 $3.00 + 0.00 0 1989 Pergamon Press plc
EFFECT OF SALINITY ON THE ACTIVITIES OF DIGESTIVE PROTEASES FROM THE TILAPIA FISH, OREOCHROMIS NILOTZCUS IN DIFFERENT CULTURE ENVIRONMENTS LEE-SHING Department
of Marine
FANG and SHU-FEN
CH~OU
Resources, National Sun Yat-sen University, Kaohsiung Republic of China. Telephone: 7-5316171, ext. 5027
80424, Taiwan,
(Received 9 November 1988) Abstract-l. During the same culture period, Oreochromis niloficus raised in sea-water grew almost 60% slower than that raised in fresh water. 2. Blood osmolarity of the sea-water specimen was 380-390 mOsm and that of the fresh water specimen was 28G-300 mOsm. 3. The potency of proteases from sea-water specimens were similar to those from fresh water specimens when assayed in high salt medium (300/w NaCl). However, it was decreased significantly when assayed in normal Tris-buffer medium. 4. The activity of the enzymes from fresh water specimens did not change significantly in a salinity range from less than 0.1360 to as high as 2000/w. 5. Analysis of function-staining electrophoresis indicated that even though the potency of the digestive proteases was not decreased, the composition of the enzymes had changed by the presence of three more isozymes. 6. This also suggested that when monitoring enzyme systems of marine organisms with respect to
osmolarity, the assay system should not be directly adapted from that of mammalian system but should be adjusted according to the physiological conditions of the organism.
0. niloticus either from freshwater or sea-water ponds. Enzymes were assayed in media of different concentrations of NaCl or sea-water. Additionally, ammonium sulphate fractionation, ultra-filtration and electrophoretic analysis were applied to detect the possible molecular modulation of these enzymes.
INTRODUCTION is a freshwater fish whose production is the highest among all cultured fishes in Taiwan. However, due to increased conflict on freshwater for aquaculture usage and limited resources available for freshwater fish culture, the farming of this fish in brackish or sea-water has been attempted and has gained some degree of economic importance. Nevertheless, tilapia raised in salt water grow significantly more slowly than those cultured in fresh water. Preliminary observation demonstrated that during a similar culture period (2-3 months), the average body weight of fresh water Oreochromis niloticus was 315 + 18.6 g (n = 10) while that of salt water ones was 116 f 19.8 (n = 12). The reason(s) why this difference occurs remains unclear. The metabolic cost for osmoregulation of a fish in salt water could not be responsible for this phenomenon as the change in metabolic rate is not quite significant after acclimation (Winberg, 1960; Nordlie, 1978; Fang, 1982). Alternatively, the reason may lie in the efficiency of a digestive enzyme working at different salinities. When a fish is cultured in sea-water, the salt content of the fluid in the digestive tract could be much higher or different from that found in freshwater held fish. Since enzymes are proteins functioning in its stereo structure the stereo, or tertiary structure is maintained partly by the ionic interactions of its surrounding medium then the activity of digestive enzymes is very likely to be altered when a fish is kept in salt water. This hypothesis was examined by studying the activity of three digestive proteases of Tilapia
MATERIALS AND METHODS Fish and enzyme preparation Live 0. niloticus were obtained from fish market. Those from freshwater farms weighed from 259 to 35Og, whilst those from salt water farms weighed 78 to 163 g. Fish were fasted for at least 24 hr before experimental use. After killing, the whole digestive tract (intestine) of specimen were biopsied and the attached fat and connective tissue removed. The tract was then weighed, cut into pieces, soaked in 4 vol of chilled Tris-HCI buffer (0.05 M, pH 7.0) or different concentrations of NaCl or sea-water medium, sonicated for 5 min in 50% duty cycle. The homogenized tissue was centrifuged at 30,OOOg for 30min at 0°C. The supematant was subsequently used for enzyme assay. All the procedures were performed at a temperature of 0-4”C. Enzyme assays Chymotrypsin. The supernatant obtained was diluted two times with double-distilled water. Thirty-five ~1 was taken out and added to 3.465ml of reaction medium which contained 0.4mM L-tyrosine ethyl ester, 90mM CaC1,‘2H,O. in 10 mM Tris-HCI buffer (IJH 7.0). The reaction was monitored at the wavelength of 234 nm using a Hitachi 60-10 UV-Vis spectrophotometer (Hsu and Wu, 1979). The reaction took place at room temperature. Trypsin. Seventy ~1 of a 50% dilution of the enzyme supernatant was added into the reaction substrate medium L
439
.,
__
LEE-SHING FANG and &U-FEN CHIOU
440
Table I. Comparison of the activities of the enzymes from freshor sea-water 0. niloficus in different reaction media
for 30 min, then centrifuged (15,OOOg, 30 min at 4’C) to collect the precipitate. The precipitate was redissolved in 7 ml of 0.05 M Tris-HCl buffer (pH 7.0), dialysed overnight against the same buffer in a 4°C cold room and then either used for enzyme assay or for ultra-filtration. An ultra-filtration membrane was set at 200 K (Toyo filter, Japan). Those molecules with a molecular weight ~200,000 da passed through the membrane while those >2~,~Oda were retained. Both of the fractions were assayed for their enzyme activity. The fraction with higher activity was further examined using disc gel electrophoresis (DGE). A 7.5% polyacrylamide system with a running condition of pH 8.3, Tris-glycine buffer, 2.5 mA per tube. was used. Immediately after DOE, gels were cut into 0.5 cm pieces, then soaked in Azocoll solution (3 mg Azocoll per ml of 0. I M, pH 7.4 phosphate buffer) to examine their proteolytic activity. Activity was assessed by measuring the release of the red-coloured chromogen into the solution. Protein concentrations of the assay solution were determined by the technique of Lowry PI al. (1951).
Enzyme activity for different experimental groups Set Group Trypsi?I df I
I AB
-zM
II BC
III CD
IV CE
VBE
II 1.2L
7 2.16
II 2.94**
14 3.2t*+
9
6
10
13
0.22
2.29
x59**
8 0.37
9 5.9a**
Chymalfypsi~
df
t
9 -1.61
2.78**
Pepsin
df I
11 2.17
11 -t.90
12 4.03”
Group A: enzymes from freshwater specimen assay in 30%. sea-
water. Group B: enzymes from freshwater specimen assay in Tris-HCI buffer. Group C: enzymes from sea-water specimen assay in 30% NaCl water. Group D: enzymes from sea-water specimen assay in 30% seawater. Group E: enzymes from sea-water specimen assay in Tris-HCI buffer.
RESULTS Serum osmoiarity
containing 0.25 mM benzoyl+arginine methyl ester (BAME), 112.5 mM CaC1,.2H,O, 12.5 mM Tris-HCI buffer @H 8.0). The total volume of the reaction solution was 3.5 ml. The reaction was monitored at 254 nm. Reaction temperature was 27°C. Pepsin. 0.5 ml of a 25% dilution of the enzyme supernatant was added to 2.5 ml of reaction substrate containing 2% casein and 0.06N HCI (PH 1.8). The reaction was allowed to proceed for 10min at 35.5”C, then stopped by adding 5.0ml of 5% Trichloroacetic acid (TCA). The solution was centrifuged and the supernatant measured s~trophotometri~lly at 280 nm. All the above enzyme assays were carried out in media of different salinities (~2~, Table I) adjusted by adding NaCl or sea-water to normal T&-buffer. Sea-water was employed to adjust the salinities in order to determine whether the change of the enzyme activity was due to the change of NaCl concentration or the presence of trace elements in the sea-water. Comparison between the activities of different experimental groups was performed by applying Student’s r-test at 95% confidence level.
The average serum osmolarity of 0. niioticus cultured in fresh water was 280-300 mOsm (n = 8). That of the sea-water fish was approximately 380-390mOsm (n = 6). Thus, osmolarity provides a good physiological index to distinguish the environmental origin of experimental fish.
The actiuity of proteases salinities
in medium of different
From Fig. 1 it can be seen that the enzyme activity of trypsin, chymotrypsin and pepsin from fresh water 0. nilotieu~ did not change signi~cantly when the reaction medium salinity was varied (0.1-2~~). This showed a remarkable stability of these enzymes in different salinities. From Table 1 (set I), group AB, it can be seen that the activity of the enzymes from fresh water 0. niloticus did not change significantly when the medium was in the salinity of 300/w, adjusted by sea-water. A comparison of Fig. 1 and Table 1 (set I) revealed that neither the salinity nor the trace elements existing in sea-water influenced the activity of these enzymes, because the enzyme activity was similar either in normal T&buffer, buffer with salinity from 0-200%~ created by NaCl and buffer with 30Ko created by sea-water.
Serum osmolarity The blood of experimental fish was immediately collected after killing. It was then clotted and the osmolarity of the serum measured by an osmometer (Wesco, 5lOOC, USA). Protein analysis of the enzymes
Ammonium sulfate was slowly introduced to the crude enzyme supematant to the desired degree of saturation (3@-70%) while stirring. The solution was allowed to stand
Table 2. Change of specific acttvity of proteases Freshwater fish
Step Crude extract Ammonium sulfate (3O-70%) Ultra filtration f>2cOR)
--Total activity (A0.D.)
Chymotrypsin
______ ._.._..
..-.
Trypsin
~~.
Specific activity (AO.D./m$)
Purification
54 92
Total protein (m& 1.4546 0.8 t 31
37.24 113.15
1.oo 3.05
26
Towprotein (m8j__ I .995 0.8064
29
0.3122
92.89
2.50
18
0.5642
-Tot;-activity (A0.D.) 30
._..
Specdic activity JAO.D.+__ 15.04 32.24
Purification
il.90
2 I2
- ~I.oo2.14
Effect of salinity on Oreochromis proteases
441
trypsin
Chymotryps
in
I
Rpsi
n
l
OI 0
I
I 25
50
Salinity
100
+
200
%
Fig. 1. The activity of proteases from freshwater 0. niloticus in the reaction medium of different salinity. Each point was the average of the measurements of two fish specimens. The influence of salt water acclimation on the enzyme activity with respect to the salinity change of reaction medium Table 1 (set IV), group CE demonstrates that the activity of the proteases from the sea-water 0. niIoticus decreased significantly when the reaction took place in normal medium (pH 7.0, 0.05 M Tris-buffer) as compared with that in 30%0 NaCl medium. It was also less potent when compared with that of the proteases from fresh water fish working in normal medium (Table 1, set V, group BE). However, the proteases of sea-water specimens working in media of high salinity (30%0 NaCl medium) were as good as the proteases from the freshwater fish working in normal
medium (Table 1, set II, group BC). These results indicated that the proteases produced in the seawater acclimated fish can function normally only in high salinity medium. Further experiments showed that (Table 1, set III, group CD) the proteases from sea-water fish had similar activity in 30%0, either adjusted by NaCl or by sea-water. This again indicated that the presence of trace elements in sea-water did not alter enzyme activity. Protein analysis of the enzymes From Table 2, it can be seen that a 2-3-fold purification could be obtained for trypsin and chymotrypsin after ammonium sulphate fractionation
from 0. niloticus after each purification
Sea-water fish Trypsin
Chymotrypsin
TOGil
Total
activity (A0.D.)
protein (w)
68 25
I .0675
I6
Specific activity (AO.D./mg)
Purification
Total activity (A0.D.)
Total protein (md)
Specific activity (AO.D./mg)
Purification
0.5225
63.70 47.85
1.00 0.75
41 41
2.315 1.045
19.20 39.23
1.00 2.04
0.2559
62.52
0.98
20
0.5118
39.08
2.04
442
LEE-SHINGFANG and SHU-FEN CHIOU
(81
-1
(A)
Fig. 2. The result of the native gel electrophoresis pattern of intestinal protein after partial purification. (A) From salt water 0. niloticus; (B) from freshwater 0. niloticus.
(3@-70%). In contrast, chymotrypsin from sea-water fish actually decreased a little bit after the fractionation. The results of uItrafiltration showed that most of the enzyme activities occurred in the unfiltrated fraction (> 200 K) (Table 2). This was very likely due to some artifacts such as clotting of the membrane, since the proteolytic enzymes from fish are usually at around 30 K (Sanchez-Chiang et al., 1987). This may also be the reason that the specific activity of the enzyme did not increase after the ultrafiltration ptocess. Proteins in the fraction with enzyme activity were further analysed by electrophoresis. Results (see Fig. 2) revealed that samples from sea-water 0. niloticus had three more bands than that of the freshwater specimen. Azocoll tests for protease activity were all positive for these bands, indicating the possibility of the presence of isozymes in sea-water fish.
DISCUSSION
In spite of the many studies on the proteases of fish which correlate activity with sex, age, season, diet composition and feeding habits (Kapoor, ef al., 1975, Stroganova and Buginova, 1969, Kawai and Ikeda, 1973, Chepik, 1964, Cockson and Bourne, 1972, Hu and Wu, 1979), there are few studies on the influence of salinity on these enzymes. Broksen and Cole (1972) studied the effects of salinity upon food absorption efficiency in Cynoscion xanthulus, quoting an unusually low value for energy absorption efficiency (72.0%) even at the optimum salinity. On the other hand, Sanchez-Chiang et al. (1987) and Squires et al. (1986) reported some salt-activating gastric proteolytic enzymes from salmon and cod, indicating that the concentration of NaCl might enhance the activity of certain proteases. Macleod
(1977) studied the effect of salinity of food intake, absorption and conversion in the rainbow trout, Salmo gairdneri, and found that there was a tendency for absorption efficiency to decrease with increasing salinity. Yet, whether the process of conversion of absorbed nutrients is affected at the biochemical level by a change in the concentration or composition of ions in different environmental salinities was not clear. The results of this study demonstrate that the proteases of freshwater 0. niloficus still function normally in a medium as high as 200% (Fig. 1). This suggests that during an acute salinity changes the adverse affect of food intake in fish (Macleod, 1977) may be a result of factors other than the ability of the alimentary canal to process food such as physiological or behavioral stress. Nevertheless, the activity of acid proteases from sardine was decreased in the presence of 200~ NaCl (Noda and Muradami. 1981). Proteases from sea-water cultured 0. niloticus were as potent as those from freshwater fish (Table 1, set II, group BC). This indicated that the slow growth of sea-water 0. niloticus was not due to the inefficiency of digestive enzymes at high salinity. However, the amino acid composition of proteases produced in the sea-water acclimated fish is very likely to be somewhat different from those of the freshwater fish. This was reflected in the observation that the activity of proteases from sea-water 0. niioticus decreased significantly when measured in normal freshwater medium, along with the presence of additional proteolytic bands on electrophoresis analysis (Table 1. set IV, group CE, Fig. 2). Certain proteins formed in high salt-acclimated bacteria arc more acidic to compensate for the ionic strength of its surrounding medium. These proteins disintegrate in low salt media (Hochachka and Somero, 1973). The digestive proteases formed in sea-water cultured 0. niloticus may have come to a similar solution to overcome the high salt in the gut. Therefore, they can maintain their activity in high salt medium but become less potent when the salt concentration is decreased. This potency change might be attributed to the change of ionic strength of the medium but not the presence of trace elements in sea-water, as the activities of the enzymes were not significantly different when the high salinity media were made from either NaCl or sea-water (Table I, set 111, group CD). Shehadah and Gordon (1969) reported that the composition of sea-water ingested by rainbow trout, Salmo gairdneri, gradually changes due to the absorption by the gut. This may influence enzyme activities but was not investigated in this study. This results presented thus demonstrate the slow growing of 0. niloticus in sea-water is not due to the inefficiency of digestive enzymes in high salinity which was compensated for by producing salt adapted isozymes. The production of these enzymes has also been observed in migrating salmon, Oncorhynchus kera, passing from estuarine to marine environments (Sgnchez-Chiang et ul., 1987). On the other hand, this study indicates that more attention should be paid to characte~stic alternations in gastro-intenstinal physiology of salt water adapted
Effect of salinity organisms. The of the reaction functionally.
osmolarity medium
on Oreochromis proteases
or composition of ions might bias the result
Acknowledgements-The authors were grateful to Dr C.-M. Kuo for valuable discussion and to Mr C.-K. Tang and Miss B.-N. Lee for technical help. This research was partly supported by a research grant (NSC-77-0209-Bl lO-02) from the National Science Council to F.L.S.
REFERENCES
Brocksen R. W. and Cole R. E. (1972) Physiological responses of three species of fishes to various salinities. J. Fish. Res. Bd Can. 29, 399405. Chepik K. (1964) Activity of digestive enzymes at different seasons of the year. Biol. Abstr. (1966) 60747. Cockson A. and Bourne D. (1972) Enzymes in the digestive tract of two species of euryhaline fish. Camp. Biochem. Physiol. 41A, 7 15-7 18. Fang L. S. (1982) Influence of salinity acclimation on routine metabolic rate pattern in different salinities of the Percoid fish, Girella nigricans. Bull. Inst. 2001. Acad. Sinica. 21, 21-26. Hsu Y. L. and Wu J. L. (1979) The relationship between feeding habits and digestive proteases of some freshwater fishes. Bull. Inst. 2001. Acad. Sinica. 18, 45-53. Hochachka P. W. and Somero G. N. (1973) Strategies of Biochemical Adaptation (Edited by Hochachka P. and Somero G.), pp. -106108: W. B. Saunders Co., London. Kaooor B. G.. Smith H. and Veriehina I. A. (1975) \ , The alimentary canal and digestion in teleosts. In Advances in Marine Biology 13, (Edited by Frederick S. and Young M.), pp. 109239. Academic Press London. Kawai S. and lkeda S. (1973) Studies on digestive enzyme of fishes. IV. Development of the digestive enzymes of
443
carp and block sea bream after hatching. Bull. Jpn Sac. Sci. Fish. 39, 877-881. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. MacLeod M. G. (1977) Effect of salinity on food intake, absorption and conversion in the rainbow trout, Salmo gairdneri. Mar. Biol. 43, 93-102. Noda M. and Murakami K. (1981) Studies of proteinases from the digestive organs of sardine. Purification and characterization of two acid proteinases from the stomach. Biochem. biophys. Acta 658, 27-32. Nordlie F. G. (1978) The influence of environmental salinity on respiratory oxygen demand in the euryhaline teleost, Ambassis interrupt. Comp. Biochem. Physiol. 59A, 271-274. Pratap K. M., Padmakar V. D. and Sudip K. B. (1978) Studies on intestinal protease; isolation, purification and effect of dietary proteins on alkaline protease activity of the air-breathing fish, Clarias batrachus (Linn). Hydrobiologia 57, 11.15. Sanchez-Chiang L., Cisternas E. and Ponce 0. (1987) Partial purification of pepsins from adult and juvenile Salmon fish Oncorhynchus Kefa. Effect of NaCl on proteolytic activities. Comp. Biochem. Physiol. 87B, 793-797. Shehadah Z. H. and Gordon M. S. (1969) The role of the intestine in salinity adaption of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 30, 397418. Squires E. J., Haard N. F. and Feltham L. A. W. (1986) Gastric proteases of Greenland cod Gadus ogac. Structural properties. Biochem. cell. Biol. 64, 215-222. Stroganova N. S. Buginova V. S. (1969) Enzymatic activity of the grass carp, Ctenopharyngodon idellus, intestinal tract. II. Proteolytic enzymes. Chem. Absrr. (1970) 72, 1078. Winberg G. G. (1960) Rate of metabolic and food requirements of fishes. Trans. Fish. Res. Bd Can. 194, I-202.
1989
0300-9629/89 $3.00 + 0.00 0 1989 Pergamon Press plc
EFFECT OF SALINITY ON THE ACTIVITIES OF DIGESTIVE PROTEASES FROM THE TILAPIA FISH, OREOCHROMIS NILOTZCUS IN DIFFERENT CULTURE ENVIRONMENTS LEE-SHING Department
of Marine
FANG and SHU-FEN
CH~OU
Resources, National Sun Yat-sen University, Kaohsiung Republic of China. Telephone: 7-5316171, ext. 5027
80424, Taiwan,
(Received 9 November 1988) Abstract-l. During the same culture period, Oreochromis niloficus raised in sea-water grew almost 60% slower than that raised in fresh water. 2. Blood osmolarity of the sea-water specimen was 380-390 mOsm and that of the fresh water specimen was 28G-300 mOsm. 3. The potency of proteases from sea-water specimens were similar to those from fresh water specimens when assayed in high salt medium (300/w NaCl). However, it was decreased significantly when assayed in normal Tris-buffer medium. 4. The activity of the enzymes from fresh water specimens did not change significantly in a salinity range from less than 0.1360 to as high as 2000/w. 5. Analysis of function-staining electrophoresis indicated that even though the potency of the digestive proteases was not decreased, the composition of the enzymes had changed by the presence of three more isozymes. 6. This also suggested that when monitoring enzyme systems of marine organisms with respect to
osmolarity, the assay system should not be directly adapted from that of mammalian system but should be adjusted according to the physiological conditions of the organism.
0. niloticus either from freshwater or sea-water ponds. Enzymes were assayed in media of different concentrations of NaCl or sea-water. Additionally, ammonium sulphate fractionation, ultra-filtration and electrophoretic analysis were applied to detect the possible molecular modulation of these enzymes.
INTRODUCTION is a freshwater fish whose production is the highest among all cultured fishes in Taiwan. However, due to increased conflict on freshwater for aquaculture usage and limited resources available for freshwater fish culture, the farming of this fish in brackish or sea-water has been attempted and has gained some degree of economic importance. Nevertheless, tilapia raised in salt water grow significantly more slowly than those cultured in fresh water. Preliminary observation demonstrated that during a similar culture period (2-3 months), the average body weight of fresh water Oreochromis niloticus was 315 + 18.6 g (n = 10) while that of salt water ones was 116 f 19.8 (n = 12). The reason(s) why this difference occurs remains unclear. The metabolic cost for osmoregulation of a fish in salt water could not be responsible for this phenomenon as the change in metabolic rate is not quite significant after acclimation (Winberg, 1960; Nordlie, 1978; Fang, 1982). Alternatively, the reason may lie in the efficiency of a digestive enzyme working at different salinities. When a fish is cultured in sea-water, the salt content of the fluid in the digestive tract could be much higher or different from that found in freshwater held fish. Since enzymes are proteins functioning in its stereo structure the stereo, or tertiary structure is maintained partly by the ionic interactions of its surrounding medium then the activity of digestive enzymes is very likely to be altered when a fish is kept in salt water. This hypothesis was examined by studying the activity of three digestive proteases of Tilapia
MATERIALS AND METHODS Fish and enzyme preparation Live 0. niloticus were obtained from fish market. Those from freshwater farms weighed from 259 to 35Og, whilst those from salt water farms weighed 78 to 163 g. Fish were fasted for at least 24 hr before experimental use. After killing, the whole digestive tract (intestine) of specimen were biopsied and the attached fat and connective tissue removed. The tract was then weighed, cut into pieces, soaked in 4 vol of chilled Tris-HCI buffer (0.05 M, pH 7.0) or different concentrations of NaCl or sea-water medium, sonicated for 5 min in 50% duty cycle. The homogenized tissue was centrifuged at 30,OOOg for 30min at 0°C. The supematant was subsequently used for enzyme assay. All the procedures were performed at a temperature of 0-4”C. Enzyme assays Chymotrypsin. The supernatant obtained was diluted two times with double-distilled water. Thirty-five ~1 was taken out and added to 3.465ml of reaction medium which contained 0.4mM L-tyrosine ethyl ester, 90mM CaC1,‘2H,O. in 10 mM Tris-HCI buffer (IJH 7.0). The reaction was monitored at the wavelength of 234 nm using a Hitachi 60-10 UV-Vis spectrophotometer (Hsu and Wu, 1979). The reaction took place at room temperature. Trypsin. Seventy ~1 of a 50% dilution of the enzyme supernatant was added into the reaction substrate medium L
439
.,
__
LEE-SHING FANG and &U-FEN CHIOU
440
Table I. Comparison of the activities of the enzymes from freshor sea-water 0. niloficus in different reaction media
for 30 min, then centrifuged (15,OOOg, 30 min at 4’C) to collect the precipitate. The precipitate was redissolved in 7 ml of 0.05 M Tris-HCl buffer (pH 7.0), dialysed overnight against the same buffer in a 4°C cold room and then either used for enzyme assay or for ultra-filtration. An ultra-filtration membrane was set at 200 K (Toyo filter, Japan). Those molecules with a molecular weight ~200,000 da passed through the membrane while those >2~,~Oda were retained. Both of the fractions were assayed for their enzyme activity. The fraction with higher activity was further examined using disc gel electrophoresis (DGE). A 7.5% polyacrylamide system with a running condition of pH 8.3, Tris-glycine buffer, 2.5 mA per tube. was used. Immediately after DOE, gels were cut into 0.5 cm pieces, then soaked in Azocoll solution (3 mg Azocoll per ml of 0. I M, pH 7.4 phosphate buffer) to examine their proteolytic activity. Activity was assessed by measuring the release of the red-coloured chromogen into the solution. Protein concentrations of the assay solution were determined by the technique of Lowry PI al. (1951).
Enzyme activity for different experimental groups Set Group Trypsi?I df I
I AB
-zM
II BC
III CD
IV CE
VBE
II 1.2L
7 2.16
II 2.94**
14 3.2t*+
9
6
10
13
0.22
2.29
x59**
8 0.37
9 5.9a**
Chymalfypsi~
df
t
9 -1.61
2.78**
Pepsin
df I
11 2.17
11 -t.90
12 4.03”
Group A: enzymes from freshwater specimen assay in 30%. sea-
water. Group B: enzymes from freshwater specimen assay in Tris-HCI buffer. Group C: enzymes from sea-water specimen assay in 30% NaCl water. Group D: enzymes from sea-water specimen assay in 30% seawater. Group E: enzymes from sea-water specimen assay in Tris-HCI buffer.
RESULTS Serum osmoiarity
containing 0.25 mM benzoyl+arginine methyl ester (BAME), 112.5 mM CaC1,.2H,O, 12.5 mM Tris-HCI buffer @H 8.0). The total volume of the reaction solution was 3.5 ml. The reaction was monitored at 254 nm. Reaction temperature was 27°C. Pepsin. 0.5 ml of a 25% dilution of the enzyme supernatant was added to 2.5 ml of reaction substrate containing 2% casein and 0.06N HCI (PH 1.8). The reaction was allowed to proceed for 10min at 35.5”C, then stopped by adding 5.0ml of 5% Trichloroacetic acid (TCA). The solution was centrifuged and the supernatant measured s~trophotometri~lly at 280 nm. All the above enzyme assays were carried out in media of different salinities (~2~, Table I) adjusted by adding NaCl or sea-water to normal T&-buffer. Sea-water was employed to adjust the salinities in order to determine whether the change of the enzyme activity was due to the change of NaCl concentration or the presence of trace elements in the sea-water. Comparison between the activities of different experimental groups was performed by applying Student’s r-test at 95% confidence level.
The average serum osmolarity of 0. niioticus cultured in fresh water was 280-300 mOsm (n = 8). That of the sea-water fish was approximately 380-390mOsm (n = 6). Thus, osmolarity provides a good physiological index to distinguish the environmental origin of experimental fish.
The actiuity of proteases salinities
in medium of different
From Fig. 1 it can be seen that the enzyme activity of trypsin, chymotrypsin and pepsin from fresh water 0. nilotieu~ did not change signi~cantly when the reaction medium salinity was varied (0.1-2~~). This showed a remarkable stability of these enzymes in different salinities. From Table 1 (set I), group AB, it can be seen that the activity of the enzymes from fresh water 0. niloticus did not change significantly when the medium was in the salinity of 300/w, adjusted by sea-water. A comparison of Fig. 1 and Table 1 (set I) revealed that neither the salinity nor the trace elements existing in sea-water influenced the activity of these enzymes, because the enzyme activity was similar either in normal T&buffer, buffer with salinity from 0-200%~ created by NaCl and buffer with 30Ko created by sea-water.
Serum osmolarity The blood of experimental fish was immediately collected after killing. It was then clotted and the osmolarity of the serum measured by an osmometer (Wesco, 5lOOC, USA). Protein analysis of the enzymes
Ammonium sulfate was slowly introduced to the crude enzyme supematant to the desired degree of saturation (3@-70%) while stirring. The solution was allowed to stand
Table 2. Change of specific acttvity of proteases Freshwater fish
Step Crude extract Ammonium sulfate (3O-70%) Ultra filtration f>2cOR)
--Total activity (A0.D.)
Chymotrypsin
______ ._.._..
..-.
Trypsin
~~.
Specific activity (AO.D./m$)
Purification
54 92
Total protein (m& 1.4546 0.8 t 31
37.24 113.15
1.oo 3.05
26
Towprotein (m8j__ I .995 0.8064
29
0.3122
92.89
2.50
18
0.5642
-Tot;-activity (A0.D.) 30
._..
Specdic activity JAO.D.+__ 15.04 32.24
Purification
il.90
2 I2
- ~I.oo2.14
Effect of salinity on Oreochromis proteases
441
trypsin
Chymotryps
in
I
Rpsi
n
l
OI 0
I
I 25
50
Salinity
100
+
200
%
Fig. 1. The activity of proteases from freshwater 0. niloticus in the reaction medium of different salinity. Each point was the average of the measurements of two fish specimens. The influence of salt water acclimation on the enzyme activity with respect to the salinity change of reaction medium Table 1 (set IV), group CE demonstrates that the activity of the proteases from the sea-water 0. niIoticus decreased significantly when the reaction took place in normal medium (pH 7.0, 0.05 M Tris-buffer) as compared with that in 30%0 NaCl medium. It was also less potent when compared with that of the proteases from fresh water fish working in normal medium (Table 1, set V, group BE). However, the proteases of sea-water specimens working in media of high salinity (30%0 NaCl medium) were as good as the proteases from the freshwater fish working in normal
medium (Table 1, set II, group BC). These results indicated that the proteases produced in the seawater acclimated fish can function normally only in high salinity medium. Further experiments showed that (Table 1, set III, group CD) the proteases from sea-water fish had similar activity in 30%0, either adjusted by NaCl or by sea-water. This again indicated that the presence of trace elements in sea-water did not alter enzyme activity. Protein analysis of the enzymes From Table 2, it can be seen that a 2-3-fold purification could be obtained for trypsin and chymotrypsin after ammonium sulphate fractionation
from 0. niloticus after each purification
Sea-water fish Trypsin
Chymotrypsin
TOGil
Total
activity (A0.D.)
protein (w)
68 25
I .0675
I6
Specific activity (AO.D./mg)
Purification
Total activity (A0.D.)
Total protein (md)
Specific activity (AO.D./mg)
Purification
0.5225
63.70 47.85
1.00 0.75
41 41
2.315 1.045
19.20 39.23
1.00 2.04
0.2559
62.52
0.98
20
0.5118
39.08
2.04
442
LEE-SHINGFANG and SHU-FEN CHIOU
(81
-1
(A)
Fig. 2. The result of the native gel electrophoresis pattern of intestinal protein after partial purification. (A) From salt water 0. niloticus; (B) from freshwater 0. niloticus.
(3@-70%). In contrast, chymotrypsin from sea-water fish actually decreased a little bit after the fractionation. The results of uItrafiltration showed that most of the enzyme activities occurred in the unfiltrated fraction (> 200 K) (Table 2). This was very likely due to some artifacts such as clotting of the membrane, since the proteolytic enzymes from fish are usually at around 30 K (Sanchez-Chiang et al., 1987). This may also be the reason that the specific activity of the enzyme did not increase after the ultrafiltration ptocess. Proteins in the fraction with enzyme activity were further analysed by electrophoresis. Results (see Fig. 2) revealed that samples from sea-water 0. niloticus had three more bands than that of the freshwater specimen. Azocoll tests for protease activity were all positive for these bands, indicating the possibility of the presence of isozymes in sea-water fish.
DISCUSSION
In spite of the many studies on the proteases of fish which correlate activity with sex, age, season, diet composition and feeding habits (Kapoor, ef al., 1975, Stroganova and Buginova, 1969, Kawai and Ikeda, 1973, Chepik, 1964, Cockson and Bourne, 1972, Hu and Wu, 1979), there are few studies on the influence of salinity on these enzymes. Broksen and Cole (1972) studied the effects of salinity upon food absorption efficiency in Cynoscion xanthulus, quoting an unusually low value for energy absorption efficiency (72.0%) even at the optimum salinity. On the other hand, Sanchez-Chiang et al. (1987) and Squires et al. (1986) reported some salt-activating gastric proteolytic enzymes from salmon and cod, indicating that the concentration of NaCl might enhance the activity of certain proteases. Macleod
(1977) studied the effect of salinity of food intake, absorption and conversion in the rainbow trout, Salmo gairdneri, and found that there was a tendency for absorption efficiency to decrease with increasing salinity. Yet, whether the process of conversion of absorbed nutrients is affected at the biochemical level by a change in the concentration or composition of ions in different environmental salinities was not clear. The results of this study demonstrate that the proteases of freshwater 0. niloficus still function normally in a medium as high as 200% (Fig. 1). This suggests that during an acute salinity changes the adverse affect of food intake in fish (Macleod, 1977) may be a result of factors other than the ability of the alimentary canal to process food such as physiological or behavioral stress. Nevertheless, the activity of acid proteases from sardine was decreased in the presence of 200~ NaCl (Noda and Muradami. 1981). Proteases from sea-water cultured 0. niloticus were as potent as those from freshwater fish (Table 1, set II, group BC). This indicated that the slow growth of sea-water 0. niloticus was not due to the inefficiency of digestive enzymes at high salinity. However, the amino acid composition of proteases produced in the sea-water acclimated fish is very likely to be somewhat different from those of the freshwater fish. This was reflected in the observation that the activity of proteases from sea-water 0. niioticus decreased significantly when measured in normal freshwater medium, along with the presence of additional proteolytic bands on electrophoresis analysis (Table 1. set IV, group CE, Fig. 2). Certain proteins formed in high salt-acclimated bacteria arc more acidic to compensate for the ionic strength of its surrounding medium. These proteins disintegrate in low salt media (Hochachka and Somero, 1973). The digestive proteases formed in sea-water cultured 0. niloticus may have come to a similar solution to overcome the high salt in the gut. Therefore, they can maintain their activity in high salt medium but become less potent when the salt concentration is decreased. This potency change might be attributed to the change of ionic strength of the medium but not the presence of trace elements in sea-water, as the activities of the enzymes were not significantly different when the high salinity media were made from either NaCl or sea-water (Table I, set 111, group CD). Shehadah and Gordon (1969) reported that the composition of sea-water ingested by rainbow trout, Salmo gairdneri, gradually changes due to the absorption by the gut. This may influence enzyme activities but was not investigated in this study. This results presented thus demonstrate the slow growing of 0. niloticus in sea-water is not due to the inefficiency of digestive enzymes in high salinity which was compensated for by producing salt adapted isozymes. The production of these enzymes has also been observed in migrating salmon, Oncorhynchus kera, passing from estuarine to marine environments (Sgnchez-Chiang et ul., 1987). On the other hand, this study indicates that more attention should be paid to characte~stic alternations in gastro-intenstinal physiology of salt water adapted
Effect of salinity organisms. The of the reaction functionally.
osmolarity medium
on Oreochromis proteases
or composition of ions might bias the result
Acknowledgements-The authors were grateful to Dr C.-M. Kuo for valuable discussion and to Mr C.-K. Tang and Miss B.-N. Lee for technical help. This research was partly supported by a research grant (NSC-77-0209-Bl lO-02) from the National Science Council to F.L.S.
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