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Metabolism in marine flatfish—II. Protein digestion in dover sole (Solea solea L.)
Comp. Biochem. Physiol. Vol. 81B,No. 1, pp. 217-222, 1985 Printed in Great Britain
0305-0491/85$3.00+0.00 © 1985PergamonPress Ltd
M E T A B O L I S M IN M A R I N E F L A T F I S H - - I I . P R O T E I N D I G E S T I O N IN D O V E R SOLE (SOLEA SOLEA L.)* J. CLARK,N. L. MACDONALD and J. R. STARK Department of Brewingand BiologicalSciences,Heriot-Watt University,Chambers Street, Edinburgh, UK (Tel: 031-225-8432)
(Received 10 August 1984) Abstract--1. The digestive tracts of adult and juvenile Dover sole were examined for protease activities. 2. A pepsin-like protease with an optimal pH value of 1.7 predominated in the stomach region, but the main endoprotease action in the foregut, midgut and hindgut regions was optimal in the range of pH 9.5-10.5 and showed good activity towards elastin orcein. 3. Experiments using synthetic substrates suggested the presence of chymotrypsin- and trypsin-like activities optimal between pH 7 and 8. Collagenase activity was also shown to exist in this pH region. 4. The presence of enzymescorresponding to carboxypeptidasesa and b and leucine aminopeptidase was indicated. 5. The possible significanceof these results to the farming of Dover sole is discussed.
INTRODUCTION
Within the last decade the culture of marine flatfish has assumed increasing importance. However, the main problem in the commercial farming of marine fish species is in the rearing of large numbers of juveniles suitable for fattening and the related difficulties in formulating adequate 'starter' or weaning diets. At present, the food for larval fish consists of living food organisms, the brine shrimp Artemia being the most commonly used species. The disadvantages of a live diet are the inconveniencein preparation, the cost and, above all, the problems of availability. If the expected expansion of hatchery operations materialises, the demand for live food will increase, resulting in increased cost and limited supplies. There is therefore a demand for an artificial diet which can be fed as early as possible during the rearing process. One of the most important components of an artificial diet for any marine carnivore, such as sole, is protein. Protein is known to act as both a nutrient for production of enzymes and muscle tissue and as an energy source. The relative growth efficiency offish is dependent on both the total quantity and the quality of the protein in the diet (Phillips, 1969; Cowey and Sargent, 1979). The essential amino acid requirements of fish have been studied by a number of workers (Halver et al., 1957; Cowey and Sargent, 1979) and it has been shown that plaice and sole require the same essential amino acids as the young rat and Pacific salmon (Cowey et al., 1970). It has more recently been shown (Cowey et al., 1974) that the amino acid composition or 'quality' of protein is critical to net protein utilization (Utne, 1979), particularly when the protein content of the diet is relatively low. In comparison with the studies on protein requirements of marine flatfish, few detailed investigations have been made on proteolytic digestive processes in
* This work was supported by grants from the S.E.R.C. (Marine Technology).
these fish. However, a wide variety of other fish have been surveyed for their content of proteolytic enzymes, including rainbow trout, Salmo gairdnerii L. (Kitamikado and Tachino, 1960), ayu, Plecoglossus attivelus L. (Tanaka et al., 1972), common carp, Cyprinus carpio L. (Onishi et al., 1973), and perch, Percafluviatilis L. (Hirji and Courtney, 1982). These investigations have involved the identification of the adult proteases or have compared enzyme activities at various stages of juvenile development. More recently the proteolytic enzymes of carnivorous, omnivorous and herbivorous species of freshwater fish have been compared (Jonas et al., 1983). Studies on marine species have included an examination of the carbohydrases and proteases of three marine flatfish (Yasunaga, 1972), experiments on enzyme development of the Black Sea bream, Acanthopagrus schlegeli Bleeker (Kawai and Ikeda, 1973), and investigations into the proteases in both adult and juvenile bass, Dicentrarchus labrax L. (Alliot et al., 1974, 1977). In the present work a study has been made of the proteolytic enzymes present in Dover sole as a first stage in understanding more of the metabolic processes and nutritional requirements of this flatfish. This study follows previous work on carbohydrate digestion in Dover sole (Clark et al., 1984) and runs in parallel with work being carried out at present on nutrition and microencapsulation. MATERIALS AND METHODS
Juvenile and adult sole samples were supplied by Seafresh Farms (Scotland) Ltd, Hunterston. 0-Group juveniles weighed between 15 and 34 g and were 50-150 mm long whereas adult fish were 2-group (average weight 400 g and length 300 mm). All fish had been continuously fed on artificial diets for at least 3 weeks (so there were no contaminating enzymes from live diets in any of the preparations).
Preparation of extracts The majority of these studies were carried out on juvenile fish using homogenates of the whole digestive tract. For 217
218
J. CLARK, N. L. MACDONALD and J. R. STARK
examination of different parts of the intestine the alimentary canal of adult fish was divided into stomach, foregut, midguL hindgut and rectum. Due to a lack of regional differentiation such divisions were arbitrary, being made on the basis of percentage of total gut length as suggested by Braber and de Groot (1973). All fish were starved for 3 hr before being killed by a blow to the head, and this was followed by deep-freezing. After partial thawing the intestine could be readily excised, weighed and homogenized with distilled water to give a 1 : 10 homogenate. Most analyses were carried out by using a further dilution of this sample to give a 1:40 homogenate. The lumen contents, present in the gut, were included in all homogenates unless otherwise stated. When required, pure gut homogenate was prepared by rinsing out an opened digestive tract with distilled water and then homogenizing the cleaned intestine.
Chemicals Unless otherwise stated, all chemicals were supplied by Sigma Chemical Co.
En2yDle assays Total proteolytic activity. Total proteolytic activity was estimated by a modification of the casein hydrolysis method of Kunitz (1947) as described by Dabrowski and Glogowski (1977). Digests consisted of 1°;i casein (0.5 ml), intestinal homogenate (0.25 ml) and buffer (0.25 ml). The buffers used were 0.1 M HC1 (pH 1.0), glycine HCI (pH 1.0 1.7), citrate-phosphate (pH 1.7 7.8), glycine NaOH (pH 7.8 10.1) and phosphate-NaOH (pH 11.2). After incubation at 3 7 C , ice-cold 5°0 trichloroacetic acid (1.5 ml) was added and the mixture left at 2 C for 30 rain. The samples were then centrifuged at 2000 g for 10 min and the absorbance of the supernatant solution recorded at 280 nm. Tyrosine was used as the standard and the units were expressed as #g tyrosine liberated/min/g protein. The protein content of the homogenate was determined by the method of Miller (1959).
method described in the Worthington En--yme Manual(1972). Elastin orcein (10 mg) was continuously rotated with buffer (1.0 ml) and 1:10 whole gut homogenate (0.1 ml). The mixture was centrifuged and the absorbance read at 590 nm against as appropriate elastin orcein blank which had been incubated for the same time at the same pH. Collagenase activity. Collagenase activity was recorded by measuring the release of L-hydroxyproline from purified bovine achilles tendon collagen substrate (Sigma type I) after digestion by juvenile sole gut homogenate enzymes. 1:40 whole gut homogenate (l ml) was incubated for 8 hr with 15 mg substrate in 0.5 ml buffer (0.2 M glycine NaOH). Digests were incubated in a 3 7 C constant temperature oven in continuously rotated microcentrifuge tubes. Following incubation, digests were subjected to 10 rain on centrifugation; 1 ml of supernatant being removed and hydrolysed for 3.5 hr in sealed test-tubes in the presence of 13 N HCL (1 ml). Tubes were evaporated to dryness in a 40'C vacuum oven and amino acid residues resuspended in 2 ml buffer (pH 6.0 7.0). Hydroxyproline was colorimetrically determined by the method of Woessner (1961), absorbance at 557 nm being calibrated against a standard curve. Detection q[' exoproteases. Tests were carried out for leucine aminopeptidase, carboxypeptidase a and carboxypeptidase b using leucine amide, hippurylphenylalanine and hippurylarginine respectively. Digests were incubated for 12 hr at 2 2 C and contained substrate (10 rag), whole-gut homogenate (0.1 ml) and buffer pH 8.0 (1 ml). The digests were spotted on Whatman No. 1 chromatography paper and developed in ethyl acetate : pyridine: water (10: 4: 3 by vol). Substrate blanks, enzyme blank and the appropriate amino acids (leucine, phenylalanine and arginine) were run as standards. A 0.2°~, solution of ninhydrin in acetone was used as spray followed by 4 min of heating at 125C to reveal the amino acids. Substrate and enzyme controls were used. RESULTS
Measurement q[ endoproteases using gelatin as suhstrate. Gelatin forms viscous solutions which are useful for assessing endoproteases. Endo-action results in rapid reduction in viscosity, which can readily be measured. Solutions containing 1~; gelatin in buffer of the appropriate pH (3 ml) were thoroughly mixed with whole gut homogenate ( 1 : 20) (0.3 ml) in an M4 U-tube viscometer. Flow times were taken at appropriate time intervals. It was found convenient to define the unit of activity as the reciprocal of the time taken for the viscosity to decrease by 10°~ of its original value. Trypsin-like acticio'. Trypsin-like activity was measured by recording enzyme hydrolysis of two substrates. Hydrolysis of l0 2 M p-toluenesulphonyl-L-arginine methyl ester (TAME) was followed at 247 nm using a modification of the method of Hummel (1959) while the method of Schwert and Takenaka (1955) was used to record hydrolysis of 10 -2 M benzoyl-Larginine ethyl ester (BAEE) at 253 rim. Both substrates were supplied by BDH chemicals and assays proceeded at room temperature (22~C) in 0.2 M citrate-phosphate buffers. Enzyme extracts for assays were four-fold dilutions of the supernatant from previously centrifuged 1:10 homogenate. Enzyme activity was determined as the change in absorbance per rain and was expressed as enzyme units per mg protein: 1 enzyme unit (IEU) being equivalent to a 0.001 change in absorbance per min. Chymotrypsin-like activity. Hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE) was used to measure chymotrypsinlike activity. BTEE (0.00107M) was prepared in 501~i methanol and activity recorded at 253 nm, as given by the method by Hummel (1959). Substrate was supplied by BDH chemicals and its hydrolysis by 1:40 supernatant homogenate proceeded at room temperature in 0.2 M citrate phosphate buffer. Activity was determined as above: results being expressed as enzyme units per mg protein. Elastase activity. Elastase activity was measured using elastin orcein (Sacher et al., 1955) by a modification of the
Hydrolysis o f casein T h e h y d r o l y s i s o f casein by w h o l e - g u t h o m o g e n a t e s o f D o v e r sole indicated three m a i n p H regions o f activity (Fig. 1). A t p H 1-2 a low level o f pepsin-like activity c o u l d be detected, b u t the m a j o r i t y o f p r o t e a s e action was at p H 7-8 a n d 9.5-10.5. A d u l t D o v e r sole w h o l e - g u t h o m o g e n a t e s s h o w e d the s a m e general
18.0] x c
16-0-
14-0o ca_ t~ 12.0 .£ _e I0.0 80 6.0 40
20 0
I"0
30
5:0
710
90
11-0
pH Fig.
I. H y d r o l y s i s o f casein by homogenates o f whole intestine (and contents) f r o m juvenile D o v e r sole.
Protein digestion in Dover sole Subsfrate:casein pH 1.3 t~......-~ 100 pH 7'0 c---o ....~ . I ~ , pH10-0 ~ .........•/,z~)..: \,,, 80 .D" / " " \ , " "tJ ",
Substrafe: casein
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219
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--~
1.0-
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.=E_ ~ o
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Foregu~
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0
r
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Hydrolysis of gelatin
M i d g u ~
The action of proteases was also compared using gelatin as substrate. In viscometric assays the action of enzymes on polymer molecules by random cleavage in the central region of the molecule results in a rapid reduction in viscosity; whereas the systematic removal of residues from one end has only a very minor effect. This therefore provided a means of testing for endoenzymic activity. At the three pH values used in the assays a reduction in the viscosity of the gelatin solution was noted, and the activities were: pH 1.3, 0.10 units; pH 7.0, 0.31 units; and pH 10.1, 1.25 units.
~-~ 0"5' ~&
20 30 t+0 50 Temperalure (°C)
Fig. 3. The effect of temperature of incubation on the rate of hydrolysis of casein at different pH values. 1'0"
._
10
0"5-
0 lO.O]
i.°1
6.0 4"0t 2"0 0
Trypsin- and chymotrypsin-like activities
2'.o
40
6'.0
8'.o
160
12.0
pH Fig. 2. Hydrolysis of casein by homogenates from different regions of the alimentary canal of adult Dover sole.
pattern of pH profile, except that the ratio of pepsinlike activity to total protease was much greater; whereas in younger fish (80 days) the acidic protease was absent (Clark and Stark, 1984). To examine hydrolysis of protein in the Dover sole gut in more detail, an adult intestine was divided into sections and the pH profiles of casein hydrolysis examined (Fig. 2). In the stomach region the acidic (pepsin-like) protease was predominant but these activities could not be detected in the other regions of the intestine. In the stomach, foregut and midgut regions the neutral (pH 7-8) and alkaline (pH 9.5-10.5) activities could be readily detected but in the hindgut these latter activities were eight times as strong as they were in the other three regions. It would therefore appear that, although Dover sole does not have a clearly defined stomach (as exists in mammals), it nevertheless produces a pepsinlike enzyme in this region of the intestine. 'Optimum' temperatures were determined under standard conditions, and it can be seen from Fig. 3 that the acidic protease had an optimum temperature of action under these conditions of about 40°C, whereas the neutral and alkaline proteases were optimal at 50°C. At this latter temperature the pepsinlike protease was only 74% as active as it was at 40°C.
The nature of the protease activities in the neutral region (pH 7-8) was examined using the artifical substrates p-toluenesulphonyl-L- arginine methyl ester (TAME) and benzoyl-L-tyrosine ethyl ester (BTEE). The results of these experiments (shown in Fig. 4) indicated an optimum pH value of 8.0 for the trypsinlike activity and pH 7.5 for the chymotrypsin-like activity. It would therefore appear that the neutral region of activity consisted largely of these two enzymes. 50.0:
H
Substrafes:BTEE TAME o--o /+0.(
.E
30'0
V 20.0
), <
10.0
04"0
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~0 pH
10.0
12.0
Fig. 4. Chymotrypsin- and trypsin-like activities measured in juvenile Dover sole intestine homogenates using benzoyl-Ltyrosine ethyl ether (BTEE) and p-toluenesulphonyl-L-arginine methyl ester (TAME).
220
J. CLARK,
N. L. MACDONALDand J. R. STARK
Table 1. Exoproteases in D o v e r sole intestine
Substrate
( R f value)
Product
(Rfvalue)
Enzyme
(0.72) ( )* (--)*
leucine phenylalanine arginine
(0.42) (0.47) (0.04)
leucine aminopeptidase earboxypeptidase a carboxypeptidase b
Leucine amide Hippurylphenylalanine Hippurylarginine
* These substrates did not give a positive reaction with ninhydrin.
100
60
Relafive Activity
60" 40
20"
0
,
6-0
,
7'0
,
,
8-0
90
1041
,
110
,
170
pH
Fig. 5. Elastase (O O) and collagenase ( O - - O ) activities in juvenile Dover sole intestine homogenates.
Collagenase and elastase activities
To investigate the high level of protease action at alkaline pH values (pH 9.5-10.5) collagenase and elastase assays were carried out to investigate their pH profiles. The results (Fig. 5) indicated that, whilst the collagenase activity may have contributed to the neutral region of activity, it did not explain the high level of activity in the alkaline region. Elastin orcein, on the other hand, was hydrolysed well at around pH l0 and, since this is a randomly dyed substrate, its hydrolysis must represent endoprotease activity. Exoproteases
The exoproteases were examined using the artificial substrates leucine amide, hippurylphenylalanine and hippurylarginine as tests for leucine aminopeptidase, carboxypeptidase a and carboxypeptidase b respectively. These were carried out as qualitative tests by paper chromatography and the results (Table 1) give a clear indication of the presence of hydrolytic activity towards all three substrates. DISCUSSION
Feeds for fish like Dover sole consist of large proportions of relatively expensive protein (as opposed to the much less expensive carbohydrate and fat components which can be fed in quantity to land animals). The utilization of dietary protein by salmonid fish has been reviewed by Pfeffer (1982) and the observation has been made that, at least in adult fish, which can be fed enzymically inert pellets, the fish must be able to synthesize the required compliment of protein-hydrolysing enzymes. Walton and Cowey (1982) have noted that salmonids, like many other
fish, have relatively high dietary requirements for both protein and essential amino acids, which in some cases are more than twice those of rat, chicken and pig. High percentages of dietary calories from protein have been quoted. For example, Phillips (1969) has suggested that a value of 70~o of the calorific requirements can be obtained from protein in trout. Furthermore, the fact that fish produce ammonia as the end product of protein metabolism indicates that they have the capacity to utilize this nutrient more efficiently than mammals or birds, which produce urea and uric acid respectively (Walton and Cowey, 1982). Peres (1979) has observed that there is always a problem of demonstrating that an enzyme in the digestive tract is truly the product of exocrine cells rather than of bacterial or cell desquamation origin. However, in the present study the objective is to ascertain the total compliment of enzymes which are available to the fish for the purposes of hydrolysis of the proteinaceous food materials. It is against this background that the lbllowing studies have been undertaken. Although there are reports on digestive proteases in many different species of fish (both marine and freshwater), there are few references on nutrition and digestion in sole. Also, the difficulties of attempting to correlate the results of studies on widely different species is further complicated by the fact that many different substrates are used by research groups in different laboratories. This problem is discussed by Dabrowski (1979). The object of this present project on Dover sole is to gain fundamental information on the digestive processes so that a more scientific approach can be made to the formulation of artificial diets for fish farmed in controlled environments. The essential amino acid requirements of sole and plaice have been examined by Cowey et al. (1970), and more recently Mackie et al. (1980) have examined the nature of feeding stimulants for juvenile Dover sole. In the first paper in this series (Clark et al., 1984) a survey was made of the carbohydrases present in the intestine of Dover sole. This study indicated that the number of enzymes capable of cleaving glycosidic bonds was limited to the amylase-maltase and chitinase-chitobiase systems; only trace amounts of other disaccharases were present. This paper therefore describes a similar study on the proteases in Dover sole. Acid protease
Using the casein hydrolysis method, the effect of various pH values on activity was studied. In all samples analysed, including adult and 0-group juvenile specimens, the profiles were of the type shown in Fig. 1, indicating three regions o f p H where a peak
221
Protein digestion in Dover sole of activity appeared. The activity at the acidic pH end of the range is typical of a pepsin-like enzyme, which is the major acid protease in many species offish (Fange and Grove, 1979). It is also of interest to note that in juvenile Dover sole intestine this acidic protease is not so active as it is in the adult intestine. Verification that this enzyme was of gastric origin was obtained by analysing homogenates from different sections of an adult Dover sole intestine. As shown in Fig. 2, the acidic protease is only detected in the extract from the stomach region, where it is by far the most predominant enzyme. Pepsin is secreted as a zymogen (pepsinogen), which is activated by the acid in the stomach region to the active form of the enzyme. Several pepsins and pepsinogens have been examined from the stomach regions of fish, including dogfish (Merret et al., 1969), bonito (Kubota and Ohnuma, 1970), rainbow trout (Kitamikado and Tachino, 1960; Twining et al., 1983) and some Japanese flatfish (Yasunaga, 1972). In these studies the optimal activity was generally in the region o f p H 2 for the pepsin activity. In the present study it has also been demonstrated that the acidic protease activity is capable of causing a reduction in the viscosity of the gelatin solution and is therefore an endoprotease. Neutral proteases From the pH-activity profile (Fig. 1) it was evident that more than one enzyme was acting in the neutralalkaline region. This was examined further by using specific substrates. The pH profiles for trypsin (using TAME as substrate) and for chymotrypsin activity (using BTEE as substrate) are shown in Fig. 4 and these indicate optimal pH values of 8.0 and 7.5 respectively. From the shape of the curves it appears that these activities contribute mainly to the neutral (pH 7-8) peak on the casein profile and only minimally to the alkaline (pH 9.5-10.5) peak. It is worth noting that the chymotrypsin peak (using BTEE) has a small 'shoulder' at pH 10, perhaps indicating that this substrate is being attacked to a minor extent by the alkaline protease. It is interesting at this stage to make comparisons with pH profiles from a variety of fish studies in other laboratories. Yasunaga (1972), working on some flatfish species ( Limanada yokohamae, Kareius bicoloratus and Paralichthys olivaceus), indicated that there was optimal activity at pH 8.0-8.5; the pH curves were all single-peak profiles. Other comparisons ofpH-activity profiles have been made by Dabrowski (1979), who cites pH curves from Salmo gairdnerii (Kitamikado and Tachino, 1960), Cyprinus carpio (Onishi etal., 1973), Carassius auratus (Jany, 1976) and Oncorhynchus tshawytscha (Croston, 1976), which all exhibit optimal pH values of 9.0 or greater in the neutralalkaline region. Furthermore, these profiles were also smooth single peaks. In contrast to this, the pH profile for protease activity in silver carp (Ragyanszki et al., 1977) indicates a peak at about pH 10.0 and a shoulder at pH 7.5. An even clearer indication of double-peak profiles was illustrated by Benitez and Tiro (1982), working on proteases of the milkfish Chanos chanos. These latter experiments indicated two clear optima at pH 7.0-7.6 and 9.5-10.0. The majority of the abovementioned experiments were carried out using casein as substrate and measuring the tyrosine released by
methods based on the procedure of Kunitz (1947). In Dover sole intestine homogenates the peak ofprotease activity at pH 7-8 was therefore accounted for mainly by the enzymes trypsin and chymotrypsin. Alkaline protease To investigate the major peak at pH 9.5-10.5, collagenase and elastase activities were considered. However, collagenase showed optimal activity at pH 7.3 (Fig. 5). Although there are fewer reports of collagenase activity, Yoshinaka et al. (1982) have assayed 19 species ofteleost for this enzyme using a pH of 7.5 and their studies indicated that, although the pancreatic tissue is diffuse in many teleosts, the collagenase does in fact originate from this tissue. Using elastin orcein as substrate the optimal activity in Dover sole intestinal homogenates was at pH 9.7, indicating that elastase possibly accounted for at least some of the alkaline protease peak produced when casein was used as substrate. Reports of elastase activity in fish include Chimaera monstrosa (Nilsson and Fange, 1969), and more recently this enzyme has also been studied in the pancreas of the catfish (Parasilurus asotus) (Yoshinaka et al., 1982). Exoproteases Whilst the three main regions of protease activity shown in Fig. 1 all exhibited endoprotease action as demonstrated by the reduction in viscosity of gelatin, it was appropriate to carry out tests for exoproteases. Carboxypeptidases a and b were present in homogenates of Dover sole as judged by the release of phenylalanine and arginine from their respective hippuryl derivatives (Table 1). Carboxypeptidases have been found in several other species (see Fange and Grove, 1979). Leucine aminopeptidase was detected by the hydrolysis of leucine amide. As pointed out by Khablynk and Proskuryakov (1983), leucine aminopeptidase in fish has received little attention compared with that of mammals. However, these authors have purified and examined the leucine aminopeptidase from the hepatopancreas of carp. CONCLUSIONS
It is concluded that, whereas Dover sole intestine is limited in its number of active carbohydrases (Clark et al., 1984), there is a full complement of proteindegrading enzymes. It must be emphasised that not all species of fish have this full complement of enzymes; for example, Jany (1976) states that the digestive system of bonefish (Carassius auratus Gibelio Bloch) contained trypsin and chymotrypsin but no pepsin, elastase or collagenase. Two aspects are therefore of particular interest for future investigations: firstly, the significance of the alkaline protease, and secondly, the exact levels of these enzymes in larvae and young, on-growing Dover sole. Following this identification of the activities present, these problems are now under investigation. Acknowledgement--The authors also wish to thank Mr S.T. Hull and his staff (Seafresh Farms Ltd) for their help. REFERENCES
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J, CLARK, N. L. MACDONALDand J. R. STARK
Alliot E., Pastoureaud A. and Trellu J. (1977) Evolution des activities enzymatiques dans le tube digestif au cours de la vie larvaire du Bar (Dieentrarchus labrax). Variations des prot6inogrammes et des zymogrammes. Actes coll. C.N.E.X.O. 4, 85 91. Benitz L. V. and Tiro L. B. (1982) Studies on the digestive proteases of the milkfish Chanos chanos. Mar. Biol. 71, 309-315. Braber L. and de Groot S. J. (1973) On the morphology of the alimentary tract of flatfish (Pleuronectiformes). J. Fish Biol. 5, 147-153. Clark J. and Stark, J. R. (1984) Development of digestive proteases in Dover sole Solea solea L. Poster No 10. International Symposium on Feeding and Nutrition in Fish, 10-13 July, Aberdeen. Clark J., McNaughton J. E. and Stark J. R. (1984) Metab, olism in marine flatfish--l. Carbohydrate digestion in Dover sole (Solea solea L.). Comp. Biochem. Physiol. 77B, 821 827. Cowey C. B. and Sargent J. R. (1979) Nutrition. In Fish Physiology (Edited by Hoar W. S., Randall D. J. and Brett J. R.), Vol. VIII, pp. 1-69. Academic Press, New York. Cowey C. B., Adron J. W. and Blair A. (1970) Studies on the nutrition of marine flatfish. The essential amino acid requirements of plaice and sole. J. mar. Biol. Assoc. U.K. 50, 87-95, Cowey C. B., Adron J. W. and Blair A. (1974) Studies on the nutrition of marine flatfish. Utilisation of various dietary proteins by plaice (Pleuronectes platessa). Br. J. Nutr. 31, 297-306. Croston B. (1960) Tryptic enzymes of Chinook salmon. Archs Biochem. Biophys. 89, 202-206. Dabrowski, K. I3. (1979) The role of proteolytic enzymes in fish digestion. In Cultivation ofFish Fry and its Live Food, pp. 102-126. European mariculture Society Special Publications No. 4. Dabrowski K. and Glogowski J. (1977) Studies on proteolytic enzymes of invertebrates constituting fish food. Hydrobiologia 52, 171-174. Fange R. and Grove D. (1979) Digestion. In Fish Physiology (Edited by Hoar W.S., Randall D.J. and Brett J.R.), Vol. VIII, pp. 161-261, Academic Press, New York. Halver J. E., Delong D. C. and Mertz E. T. (1957) Nutrition of salmonid fishes V. Classification of essential amino acids for Chinook salmon. J. Nutr. 63, 95-105. Hirji K. N. and Courtney W. A. M, (1982) Leucine aminopeptidase activity in the digestive tract of perch (Perca fluviatilis L.). J. Fish Biol. 21, 615-622. Hummel B. C. W. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399. Jany K. D. (1976) Studies on the digestive enzymes of the stomachless bonefish Carassius auratus Gibelio Bloch: endopeptidases. Comp. Bioehem. Physiol. 53B, 31-38. Jonas E., Ragyanszki M., Olah J. and Borass L. (1983) Proteolytic digestive enzymes of carnivorous (Silurus glanis L.), herbivorous (Hypophthalmichthys molitrix Val.) and omnivorous (Cyprinus carpio L.) fishes. Aquaculture 30, 145-154. Kawai S. and Ikeda S. (1973) Studies on digestive enzymes of fishes. IV Development of the digestive enzymes of carp and black sea bream after hatching. Bull..lap. Soc. scient. Fish. 39, 877-881. Khablynk V. V. and Proskuryakov M. T. (1983) Purification and some properties of leucine aminopepetidase from carp hepatopancreas. Prikl. Biokhim. Mikrobiol. 19, 427-430. Kitamikado M. and Tachino S. (1960) Studies on the digestive enzymes of rainbow trout, I. Proteases. Bull..lap.
Soc. scient. Fish. 26, 685 690. Kubota M. and Ohnuma A. (1970) Studies on bonito pepsin. II Enzymic properties of bonito pepsin. Bull. ,lap. Soc. scient. Fish. 36, 1152 1156. Kunitz M. (1947) Crystalline soybean trypsin inhibitor. II General properties. J. gen, Physiol. 30, 291-310. Mackie A. M., Adron J. W. and Grant P. T. (1980) Chemical nature of feeding stimulants for the juvenile Dover sole, Solea solea (L.). J. Fish Biol. 16, 701-708. Merret T. G., Bar-Eli E. and Van Vunakis, H. (1969) Pepsinogens A, C and D from smooth dogfish. Biochemistry 8, 3696-3702. Miller G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31,964. Nilsson A. and Fange R. (1969) Digestive proteases in the holocephalian fish, Chimaera monstrosa (L.). Comp, Biochem. Physiol. 31, 147 165. Onishi T., Murayama S. and Takeuchi, N. (1973) Sequence of digestive enzyme levels in carp after feeding. I I Protease in activated and zymogen forms of intestine, hepatopancrease, gall bladder and spleen. Bull. Tokai Reg. Fish. Res. Lab. 75, 33-38. Peres G. (1979) Enzymologie digestive 1. Les prot6ases, l'amylase, les enzymes chitinolytiques, les laminarinases. In Nutrition des Poissons (Edited by Fontaine M.), pp. 55-67. Actes du collague C.N.E.R.N.A., Paris. Pfeffer E. (1982) Utilisation of dietary protein by salmonid fish. Comp. Bioehem. Physiol. 73B, 51-57. Phillips A.M. (1969) Nutrition, digestion and energy utilization. In pLishPhysiology (Edited by Hoar W. S. and Randall D. J.), Vol. 1, pp. 391-432. Academic Press, London. Ragyanszki M., Jonas E., Olah J. and Boross L. (1977) Studies on proteolytic enzymes of silver carp, ttypophthalmichthys molitrix. International Seminary on Fish Nutrition and Diet Development, 19 24 September, Szarvas, Hungary. Sachar L. A., Winter K. K., Sicher N. and Frankel S. (1955) Photometric method for estimation of elastase activity. Proc. Soc. exp. Biol. Med. 90, 323. Schwert G. W. and Takenaka Y. (1965) A spectrophotometric determination of trypsin and chymotrypsin. Biochim. biophys. Acta 16, 57(~575. Tanaka M., Kawai S. and Yamarnoto S. (1972) On the development of the digestive system and changes in activities of digestive enzymes during larval and juvenile stages in Ayu. Bull. Jap. Soc. scient. Fish. 38, 1143-1152. Twining S. S., Alexander P. A., Huibregtse K. and Glick D. M, (1983) A pepsinogen from rainbow trout. Comp. Biochem. Physiol. 75B, 109 112. Utne F. (1979) Standard methods and terminology in finfish nutrition. In Proceedings of the World Symposium on Finfish Nutrition and Fishfeed Technology, 20-23 June 1978, Hamburg, pp, 437~,44. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B, 59-79. Woessner J. F. (1961) The determination of hydroxyprolinc in tissue and protein samples containing small amounts of this imino acid. Archs Biochem. Biophys. 93, 44~447. Worthington Enzyme Manual (1972) Worthington Biochemical Corp., Freehold, N J. Yasunaga Y. (1972) Studies on the digestive function of enzymes: protease and amylase of some flatfish species. Bull. Tokai Res. Fish. Res. Lab. 71, 169-175. Yoshinaka R., Tanaka H., Sato M. and Ikeda S. (1982) Purification and some properties of elastase from the pancrease of catfish. Bull. Jap. Soc. scient. Fish. 48, 573 579.
0305-0491/85$3.00+0.00 © 1985PergamonPress Ltd
M E T A B O L I S M IN M A R I N E F L A T F I S H - - I I . P R O T E I N D I G E S T I O N IN D O V E R SOLE (SOLEA SOLEA L.)* J. CLARK,N. L. MACDONALD and J. R. STARK Department of Brewingand BiologicalSciences,Heriot-Watt University,Chambers Street, Edinburgh, UK (Tel: 031-225-8432)
(Received 10 August 1984) Abstract--1. The digestive tracts of adult and juvenile Dover sole were examined for protease activities. 2. A pepsin-like protease with an optimal pH value of 1.7 predominated in the stomach region, but the main endoprotease action in the foregut, midgut and hindgut regions was optimal in the range of pH 9.5-10.5 and showed good activity towards elastin orcein. 3. Experiments using synthetic substrates suggested the presence of chymotrypsin- and trypsin-like activities optimal between pH 7 and 8. Collagenase activity was also shown to exist in this pH region. 4. The presence of enzymescorresponding to carboxypeptidasesa and b and leucine aminopeptidase was indicated. 5. The possible significanceof these results to the farming of Dover sole is discussed.
INTRODUCTION
Within the last decade the culture of marine flatfish has assumed increasing importance. However, the main problem in the commercial farming of marine fish species is in the rearing of large numbers of juveniles suitable for fattening and the related difficulties in formulating adequate 'starter' or weaning diets. At present, the food for larval fish consists of living food organisms, the brine shrimp Artemia being the most commonly used species. The disadvantages of a live diet are the inconveniencein preparation, the cost and, above all, the problems of availability. If the expected expansion of hatchery operations materialises, the demand for live food will increase, resulting in increased cost and limited supplies. There is therefore a demand for an artificial diet which can be fed as early as possible during the rearing process. One of the most important components of an artificial diet for any marine carnivore, such as sole, is protein. Protein is known to act as both a nutrient for production of enzymes and muscle tissue and as an energy source. The relative growth efficiency offish is dependent on both the total quantity and the quality of the protein in the diet (Phillips, 1969; Cowey and Sargent, 1979). The essential amino acid requirements of fish have been studied by a number of workers (Halver et al., 1957; Cowey and Sargent, 1979) and it has been shown that plaice and sole require the same essential amino acids as the young rat and Pacific salmon (Cowey et al., 1970). It has more recently been shown (Cowey et al., 1974) that the amino acid composition or 'quality' of protein is critical to net protein utilization (Utne, 1979), particularly when the protein content of the diet is relatively low. In comparison with the studies on protein requirements of marine flatfish, few detailed investigations have been made on proteolytic digestive processes in
* This work was supported by grants from the S.E.R.C. (Marine Technology).
these fish. However, a wide variety of other fish have been surveyed for their content of proteolytic enzymes, including rainbow trout, Salmo gairdnerii L. (Kitamikado and Tachino, 1960), ayu, Plecoglossus attivelus L. (Tanaka et al., 1972), common carp, Cyprinus carpio L. (Onishi et al., 1973), and perch, Percafluviatilis L. (Hirji and Courtney, 1982). These investigations have involved the identification of the adult proteases or have compared enzyme activities at various stages of juvenile development. More recently the proteolytic enzymes of carnivorous, omnivorous and herbivorous species of freshwater fish have been compared (Jonas et al., 1983). Studies on marine species have included an examination of the carbohydrases and proteases of three marine flatfish (Yasunaga, 1972), experiments on enzyme development of the Black Sea bream, Acanthopagrus schlegeli Bleeker (Kawai and Ikeda, 1973), and investigations into the proteases in both adult and juvenile bass, Dicentrarchus labrax L. (Alliot et al., 1974, 1977). In the present work a study has been made of the proteolytic enzymes present in Dover sole as a first stage in understanding more of the metabolic processes and nutritional requirements of this flatfish. This study follows previous work on carbohydrate digestion in Dover sole (Clark et al., 1984) and runs in parallel with work being carried out at present on nutrition and microencapsulation. MATERIALS AND METHODS
Juvenile and adult sole samples were supplied by Seafresh Farms (Scotland) Ltd, Hunterston. 0-Group juveniles weighed between 15 and 34 g and were 50-150 mm long whereas adult fish were 2-group (average weight 400 g and length 300 mm). All fish had been continuously fed on artificial diets for at least 3 weeks (so there were no contaminating enzymes from live diets in any of the preparations).
Preparation of extracts The majority of these studies were carried out on juvenile fish using homogenates of the whole digestive tract. For 217
218
J. CLARK, N. L. MACDONALD and J. R. STARK
examination of different parts of the intestine the alimentary canal of adult fish was divided into stomach, foregut, midguL hindgut and rectum. Due to a lack of regional differentiation such divisions were arbitrary, being made on the basis of percentage of total gut length as suggested by Braber and de Groot (1973). All fish were starved for 3 hr before being killed by a blow to the head, and this was followed by deep-freezing. After partial thawing the intestine could be readily excised, weighed and homogenized with distilled water to give a 1 : 10 homogenate. Most analyses were carried out by using a further dilution of this sample to give a 1:40 homogenate. The lumen contents, present in the gut, were included in all homogenates unless otherwise stated. When required, pure gut homogenate was prepared by rinsing out an opened digestive tract with distilled water and then homogenizing the cleaned intestine.
Chemicals Unless otherwise stated, all chemicals were supplied by Sigma Chemical Co.
En2yDle assays Total proteolytic activity. Total proteolytic activity was estimated by a modification of the casein hydrolysis method of Kunitz (1947) as described by Dabrowski and Glogowski (1977). Digests consisted of 1°;i casein (0.5 ml), intestinal homogenate (0.25 ml) and buffer (0.25 ml). The buffers used were 0.1 M HC1 (pH 1.0), glycine HCI (pH 1.0 1.7), citrate-phosphate (pH 1.7 7.8), glycine NaOH (pH 7.8 10.1) and phosphate-NaOH (pH 11.2). After incubation at 3 7 C , ice-cold 5°0 trichloroacetic acid (1.5 ml) was added and the mixture left at 2 C for 30 rain. The samples were then centrifuged at 2000 g for 10 min and the absorbance of the supernatant solution recorded at 280 nm. Tyrosine was used as the standard and the units were expressed as #g tyrosine liberated/min/g protein. The protein content of the homogenate was determined by the method of Miller (1959).
method described in the Worthington En--yme Manual(1972). Elastin orcein (10 mg) was continuously rotated with buffer (1.0 ml) and 1:10 whole gut homogenate (0.1 ml). The mixture was centrifuged and the absorbance read at 590 nm against as appropriate elastin orcein blank which had been incubated for the same time at the same pH. Collagenase activity. Collagenase activity was recorded by measuring the release of L-hydroxyproline from purified bovine achilles tendon collagen substrate (Sigma type I) after digestion by juvenile sole gut homogenate enzymes. 1:40 whole gut homogenate (l ml) was incubated for 8 hr with 15 mg substrate in 0.5 ml buffer (0.2 M glycine NaOH). Digests were incubated in a 3 7 C constant temperature oven in continuously rotated microcentrifuge tubes. Following incubation, digests were subjected to 10 rain on centrifugation; 1 ml of supernatant being removed and hydrolysed for 3.5 hr in sealed test-tubes in the presence of 13 N HCL (1 ml). Tubes were evaporated to dryness in a 40'C vacuum oven and amino acid residues resuspended in 2 ml buffer (pH 6.0 7.0). Hydroxyproline was colorimetrically determined by the method of Woessner (1961), absorbance at 557 nm being calibrated against a standard curve. Detection q[' exoproteases. Tests were carried out for leucine aminopeptidase, carboxypeptidase a and carboxypeptidase b using leucine amide, hippurylphenylalanine and hippurylarginine respectively. Digests were incubated for 12 hr at 2 2 C and contained substrate (10 rag), whole-gut homogenate (0.1 ml) and buffer pH 8.0 (1 ml). The digests were spotted on Whatman No. 1 chromatography paper and developed in ethyl acetate : pyridine: water (10: 4: 3 by vol). Substrate blanks, enzyme blank and the appropriate amino acids (leucine, phenylalanine and arginine) were run as standards. A 0.2°~, solution of ninhydrin in acetone was used as spray followed by 4 min of heating at 125C to reveal the amino acids. Substrate and enzyme controls were used. RESULTS
Measurement q[ endoproteases using gelatin as suhstrate. Gelatin forms viscous solutions which are useful for assessing endoproteases. Endo-action results in rapid reduction in viscosity, which can readily be measured. Solutions containing 1~; gelatin in buffer of the appropriate pH (3 ml) were thoroughly mixed with whole gut homogenate ( 1 : 20) (0.3 ml) in an M4 U-tube viscometer. Flow times were taken at appropriate time intervals. It was found convenient to define the unit of activity as the reciprocal of the time taken for the viscosity to decrease by 10°~ of its original value. Trypsin-like acticio'. Trypsin-like activity was measured by recording enzyme hydrolysis of two substrates. Hydrolysis of l0 2 M p-toluenesulphonyl-L-arginine methyl ester (TAME) was followed at 247 nm using a modification of the method of Hummel (1959) while the method of Schwert and Takenaka (1955) was used to record hydrolysis of 10 -2 M benzoyl-Larginine ethyl ester (BAEE) at 253 rim. Both substrates were supplied by BDH chemicals and assays proceeded at room temperature (22~C) in 0.2 M citrate-phosphate buffers. Enzyme extracts for assays were four-fold dilutions of the supernatant from previously centrifuged 1:10 homogenate. Enzyme activity was determined as the change in absorbance per rain and was expressed as enzyme units per mg protein: 1 enzyme unit (IEU) being equivalent to a 0.001 change in absorbance per min. Chymotrypsin-like activity. Hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE) was used to measure chymotrypsinlike activity. BTEE (0.00107M) was prepared in 501~i methanol and activity recorded at 253 nm, as given by the method by Hummel (1959). Substrate was supplied by BDH chemicals and its hydrolysis by 1:40 supernatant homogenate proceeded at room temperature in 0.2 M citrate phosphate buffer. Activity was determined as above: results being expressed as enzyme units per mg protein. Elastase activity. Elastase activity was measured using elastin orcein (Sacher et al., 1955) by a modification of the
Hydrolysis o f casein T h e h y d r o l y s i s o f casein by w h o l e - g u t h o m o g e n a t e s o f D o v e r sole indicated three m a i n p H regions o f activity (Fig. 1). A t p H 1-2 a low level o f pepsin-like activity c o u l d be detected, b u t the m a j o r i t y o f p r o t e a s e action was at p H 7-8 a n d 9.5-10.5. A d u l t D o v e r sole w h o l e - g u t h o m o g e n a t e s s h o w e d the s a m e general
18.0] x c
16-0-
14-0o ca_ t~ 12.0 .£ _e I0.0 80 6.0 40
20 0
I"0
30
5:0
710
90
11-0
pH Fig.
I. H y d r o l y s i s o f casein by homogenates o f whole intestine (and contents) f r o m juvenile D o v e r sole.
Protein digestion in Dover sole Subsfrate:casein pH 1.3 t~......-~ 100 pH 7'0 c---o ....~ . I ~ , pH10-0 ~ .........•/,z~)..: \,,, 80 .D" / " " \ , " "tJ ",
Substrafe: casein
1.5-
219
1,00.5" ×
0
--~
1.0-
202
.=E_ ~ o
0
Foregu~
o
0
r
60
r
Hydrolysis of gelatin
M i d g u ~
The action of proteases was also compared using gelatin as substrate. In viscometric assays the action of enzymes on polymer molecules by random cleavage in the central region of the molecule results in a rapid reduction in viscosity; whereas the systematic removal of residues from one end has only a very minor effect. This therefore provided a means of testing for endoenzymic activity. At the three pH values used in the assays a reduction in the viscosity of the gelatin solution was noted, and the activities were: pH 1.3, 0.10 units; pH 7.0, 0.31 units; and pH 10.1, 1.25 units.
~-~ 0"5' ~&
20 30 t+0 50 Temperalure (°C)
Fig. 3. The effect of temperature of incubation on the rate of hydrolysis of casein at different pH values. 1'0"
._
10
0"5-
0 lO.O]
i.°1
6.0 4"0t 2"0 0
Trypsin- and chymotrypsin-like activities
2'.o
40
6'.0
8'.o
160
12.0
pH Fig. 2. Hydrolysis of casein by homogenates from different regions of the alimentary canal of adult Dover sole.
pattern of pH profile, except that the ratio of pepsinlike activity to total protease was much greater; whereas in younger fish (80 days) the acidic protease was absent (Clark and Stark, 1984). To examine hydrolysis of protein in the Dover sole gut in more detail, an adult intestine was divided into sections and the pH profiles of casein hydrolysis examined (Fig. 2). In the stomach region the acidic (pepsin-like) protease was predominant but these activities could not be detected in the other regions of the intestine. In the stomach, foregut and midgut regions the neutral (pH 7-8) and alkaline (pH 9.5-10.5) activities could be readily detected but in the hindgut these latter activities were eight times as strong as they were in the other three regions. It would therefore appear that, although Dover sole does not have a clearly defined stomach (as exists in mammals), it nevertheless produces a pepsinlike enzyme in this region of the intestine. 'Optimum' temperatures were determined under standard conditions, and it can be seen from Fig. 3 that the acidic protease had an optimum temperature of action under these conditions of about 40°C, whereas the neutral and alkaline proteases were optimal at 50°C. At this latter temperature the pepsinlike protease was only 74% as active as it was at 40°C.
The nature of the protease activities in the neutral region (pH 7-8) was examined using the artifical substrates p-toluenesulphonyl-L- arginine methyl ester (TAME) and benzoyl-L-tyrosine ethyl ester (BTEE). The results of these experiments (shown in Fig. 4) indicated an optimum pH value of 8.0 for the trypsinlike activity and pH 7.5 for the chymotrypsin-like activity. It would therefore appear that the neutral region of activity consisted largely of these two enzymes. 50.0:
H
Substrafes:BTEE TAME o--o /+0.(
.E
30'0
V 20.0
), <
10.0
04"0
6'0
~0 pH
10.0
12.0
Fig. 4. Chymotrypsin- and trypsin-like activities measured in juvenile Dover sole intestine homogenates using benzoyl-Ltyrosine ethyl ether (BTEE) and p-toluenesulphonyl-L-arginine methyl ester (TAME).
220
J. CLARK,
N. L. MACDONALDand J. R. STARK
Table 1. Exoproteases in D o v e r sole intestine
Substrate
( R f value)
Product
(Rfvalue)
Enzyme
(0.72) ( )* (--)*
leucine phenylalanine arginine
(0.42) (0.47) (0.04)
leucine aminopeptidase earboxypeptidase a carboxypeptidase b
Leucine amide Hippurylphenylalanine Hippurylarginine
* These substrates did not give a positive reaction with ninhydrin.
100
60
Relafive Activity
60" 40
20"
0
,
6-0
,
7'0
,
,
8-0
90
1041
,
110
,
170
pH
Fig. 5. Elastase (O O) and collagenase ( O - - O ) activities in juvenile Dover sole intestine homogenates.
Collagenase and elastase activities
To investigate the high level of protease action at alkaline pH values (pH 9.5-10.5) collagenase and elastase assays were carried out to investigate their pH profiles. The results (Fig. 5) indicated that, whilst the collagenase activity may have contributed to the neutral region of activity, it did not explain the high level of activity in the alkaline region. Elastin orcein, on the other hand, was hydrolysed well at around pH l0 and, since this is a randomly dyed substrate, its hydrolysis must represent endoprotease activity. Exoproteases
The exoproteases were examined using the artificial substrates leucine amide, hippurylphenylalanine and hippurylarginine as tests for leucine aminopeptidase, carboxypeptidase a and carboxypeptidase b respectively. These were carried out as qualitative tests by paper chromatography and the results (Table 1) give a clear indication of the presence of hydrolytic activity towards all three substrates. DISCUSSION
Feeds for fish like Dover sole consist of large proportions of relatively expensive protein (as opposed to the much less expensive carbohydrate and fat components which can be fed in quantity to land animals). The utilization of dietary protein by salmonid fish has been reviewed by Pfeffer (1982) and the observation has been made that, at least in adult fish, which can be fed enzymically inert pellets, the fish must be able to synthesize the required compliment of protein-hydrolysing enzymes. Walton and Cowey (1982) have noted that salmonids, like many other
fish, have relatively high dietary requirements for both protein and essential amino acids, which in some cases are more than twice those of rat, chicken and pig. High percentages of dietary calories from protein have been quoted. For example, Phillips (1969) has suggested that a value of 70~o of the calorific requirements can be obtained from protein in trout. Furthermore, the fact that fish produce ammonia as the end product of protein metabolism indicates that they have the capacity to utilize this nutrient more efficiently than mammals or birds, which produce urea and uric acid respectively (Walton and Cowey, 1982). Peres (1979) has observed that there is always a problem of demonstrating that an enzyme in the digestive tract is truly the product of exocrine cells rather than of bacterial or cell desquamation origin. However, in the present study the objective is to ascertain the total compliment of enzymes which are available to the fish for the purposes of hydrolysis of the proteinaceous food materials. It is against this background that the lbllowing studies have been undertaken. Although there are reports on digestive proteases in many different species of fish (both marine and freshwater), there are few references on nutrition and digestion in sole. Also, the difficulties of attempting to correlate the results of studies on widely different species is further complicated by the fact that many different substrates are used by research groups in different laboratories. This problem is discussed by Dabrowski (1979). The object of this present project on Dover sole is to gain fundamental information on the digestive processes so that a more scientific approach can be made to the formulation of artificial diets for fish farmed in controlled environments. The essential amino acid requirements of sole and plaice have been examined by Cowey et al. (1970), and more recently Mackie et al. (1980) have examined the nature of feeding stimulants for juvenile Dover sole. In the first paper in this series (Clark et al., 1984) a survey was made of the carbohydrases present in the intestine of Dover sole. This study indicated that the number of enzymes capable of cleaving glycosidic bonds was limited to the amylase-maltase and chitinase-chitobiase systems; only trace amounts of other disaccharases were present. This paper therefore describes a similar study on the proteases in Dover sole. Acid protease
Using the casein hydrolysis method, the effect of various pH values on activity was studied. In all samples analysed, including adult and 0-group juvenile specimens, the profiles were of the type shown in Fig. 1, indicating three regions o f p H where a peak
221
Protein digestion in Dover sole of activity appeared. The activity at the acidic pH end of the range is typical of a pepsin-like enzyme, which is the major acid protease in many species offish (Fange and Grove, 1979). It is also of interest to note that in juvenile Dover sole intestine this acidic protease is not so active as it is in the adult intestine. Verification that this enzyme was of gastric origin was obtained by analysing homogenates from different sections of an adult Dover sole intestine. As shown in Fig. 2, the acidic protease is only detected in the extract from the stomach region, where it is by far the most predominant enzyme. Pepsin is secreted as a zymogen (pepsinogen), which is activated by the acid in the stomach region to the active form of the enzyme. Several pepsins and pepsinogens have been examined from the stomach regions of fish, including dogfish (Merret et al., 1969), bonito (Kubota and Ohnuma, 1970), rainbow trout (Kitamikado and Tachino, 1960; Twining et al., 1983) and some Japanese flatfish (Yasunaga, 1972). In these studies the optimal activity was generally in the region o f p H 2 for the pepsin activity. In the present study it has also been demonstrated that the acidic protease activity is capable of causing a reduction in the viscosity of the gelatin solution and is therefore an endoprotease. Neutral proteases From the pH-activity profile (Fig. 1) it was evident that more than one enzyme was acting in the neutralalkaline region. This was examined further by using specific substrates. The pH profiles for trypsin (using TAME as substrate) and for chymotrypsin activity (using BTEE as substrate) are shown in Fig. 4 and these indicate optimal pH values of 8.0 and 7.5 respectively. From the shape of the curves it appears that these activities contribute mainly to the neutral (pH 7-8) peak on the casein profile and only minimally to the alkaline (pH 9.5-10.5) peak. It is worth noting that the chymotrypsin peak (using BTEE) has a small 'shoulder' at pH 10, perhaps indicating that this substrate is being attacked to a minor extent by the alkaline protease. It is interesting at this stage to make comparisons with pH profiles from a variety of fish studies in other laboratories. Yasunaga (1972), working on some flatfish species ( Limanada yokohamae, Kareius bicoloratus and Paralichthys olivaceus), indicated that there was optimal activity at pH 8.0-8.5; the pH curves were all single-peak profiles. Other comparisons ofpH-activity profiles have been made by Dabrowski (1979), who cites pH curves from Salmo gairdnerii (Kitamikado and Tachino, 1960), Cyprinus carpio (Onishi etal., 1973), Carassius auratus (Jany, 1976) and Oncorhynchus tshawytscha (Croston, 1976), which all exhibit optimal pH values of 9.0 or greater in the neutralalkaline region. Furthermore, these profiles were also smooth single peaks. In contrast to this, the pH profile for protease activity in silver carp (Ragyanszki et al., 1977) indicates a peak at about pH 10.0 and a shoulder at pH 7.5. An even clearer indication of double-peak profiles was illustrated by Benitez and Tiro (1982), working on proteases of the milkfish Chanos chanos. These latter experiments indicated two clear optima at pH 7.0-7.6 and 9.5-10.0. The majority of the abovementioned experiments were carried out using casein as substrate and measuring the tyrosine released by
methods based on the procedure of Kunitz (1947). In Dover sole intestine homogenates the peak ofprotease activity at pH 7-8 was therefore accounted for mainly by the enzymes trypsin and chymotrypsin. Alkaline protease To investigate the major peak at pH 9.5-10.5, collagenase and elastase activities were considered. However, collagenase showed optimal activity at pH 7.3 (Fig. 5). Although there are fewer reports of collagenase activity, Yoshinaka et al. (1982) have assayed 19 species ofteleost for this enzyme using a pH of 7.5 and their studies indicated that, although the pancreatic tissue is diffuse in many teleosts, the collagenase does in fact originate from this tissue. Using elastin orcein as substrate the optimal activity in Dover sole intestinal homogenates was at pH 9.7, indicating that elastase possibly accounted for at least some of the alkaline protease peak produced when casein was used as substrate. Reports of elastase activity in fish include Chimaera monstrosa (Nilsson and Fange, 1969), and more recently this enzyme has also been studied in the pancreas of the catfish (Parasilurus asotus) (Yoshinaka et al., 1982). Exoproteases Whilst the three main regions of protease activity shown in Fig. 1 all exhibited endoprotease action as demonstrated by the reduction in viscosity of gelatin, it was appropriate to carry out tests for exoproteases. Carboxypeptidases a and b were present in homogenates of Dover sole as judged by the release of phenylalanine and arginine from their respective hippuryl derivatives (Table 1). Carboxypeptidases have been found in several other species (see Fange and Grove, 1979). Leucine aminopeptidase was detected by the hydrolysis of leucine amide. As pointed out by Khablynk and Proskuryakov (1983), leucine aminopeptidase in fish has received little attention compared with that of mammals. However, these authors have purified and examined the leucine aminopeptidase from the hepatopancreas of carp. CONCLUSIONS
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