Leucine aminopeptidase in the digestive tract of Dover sole [Solea solea (L.)]

Aquaculture, 61(1987) 231-239 Elsevier Science Publishers B.V., Amsterdam

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Leucine Aminopeptidase in the Digestive Tract of Dover Sole [Solea solea ( L. ) ] J. CLARK, N. L. MACDONALD

and J. R. STARK

Department of Brewing and Biological Sciences, Heriot- Watt University, Edinburgh (Great Britain) (Accepted

1 December

1986)

ABSTRACT Clark, J., MacDonald, N. L. and Stark, J. R., 1987. Leucine aminopeptidase of Dover sole [ Solea solea (L.)] . Aquaculture, 61: 231-239.

in the digestive tract

Leucine aminopeptidase from the digestive tract of Dover sole has been shown to have maximum activity at pH 8.3 and at 50°C. The enzyme is activated by manganese ions and inhibited by EDTA. The rate of hydrolysis of leucinamide with uncentrifuged homogenate is four times as fast as when clear supernatant extracts are used. Of the total intestinal leucine aminopeptidase, 42% is present in the midgut, 33% in the foregut and the remainder distributed between stomach, hindgut and rectum. In a study of the effect of inhibitors,TPCK was the most effective, indicating that histidine is probably involved at the active centre.

INTRODUCTION

Leucine aminopeptidase (LAP, E.C. 3.4.11.1) is classified as a zinc-metalloproteinase which hydrolyses single amino acids from The N-terminus of peptide chains. It shows overlapping specificity with the microsomal enzyme, a! aminoacyl peptide hydrolase (E.C. 3.4.11.2)) but leucine aminopeptidase is distinguishable in that it originates from the cytosol and shows preference for the hydrolysis of peptide bonds involving L-leucine. Leucine aminopeptidase activity was first reported in extracts of hog intestinal mucosa (LinderstromLang, 1929) and similar activity has subsequently been identified in several mammalian species, plants and microorganisms. While the presence of activity in intestinal extracts suggests a digestive function, leucine aminopeptidase is widely distributed in mammalian tisues, occurring in extracts of lung, spleen, kidney, cardiac muscle and serum (Hafkenscheid, 1984). The most commonly used substrate for its detection is leucinamide, the hydrolysis of which can be measured spectrophotometrically or titrimetrically. In comparison with the large volume of literature on the endoproteases such

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as trypsin and chymotrypsin, research into leucine aminopeptidase in fish digestion has received much less attention. A limited number of reports suggest that LAP may be widespread in the digestive tracts of both marine and freshwater fish. For example, LAP has been reported in the digestive tract of marine species such as cod (Overnell, 1973) and bass (Alliot et al., 1977) while similar activity has been examined in freshwater fish such as perch (Hirji and Courtney, 1982) and carp (Khablyuk and Proskuryakov, 1983a, b) . Previous work on the development of proteases in Dover sole (Clark et al., 1986) indicated that leucine aminopeptidase activity in whole gut homogenates increased with the maturity of the fish from 24 to 200 days and then showed a slight decrease. Protein, lipid and carbohydrate are all sources of metabolic energy in fish diets. However, the protein component of the diet in carnivorous marine fish is catabolised to a greater degree than in warm blooded animals. Although the non-protein energy content of the diet can be increased in order to ‘spare’ protein for growth purpose, it is also important that the ‘quality’ of dietary protein is such that it is efficiently digested and absorbed in the gut. Therefore, in order to process efficient diets for Dover sole culture there is a need to match the dietary protein content with the digestive capability of the fish. Protein digestion in fish and mammalian species involves hydrolysis by both endo- and exoproteases. While endoproteases cause the initial hydrolysis of proteins to peptides, sequential cleavage of peptides by exoproteases such as LAP is equally important in that it ensures the availability of free amino acids which can be readily absorbed from the gut lumen. In the present study the leucine aminopeptidase of Dover sole has been examined as part of an ongoing programme of research to examine the digestive enzymes in selected species of marine flatfish. MATERIALS

AND METHODS

Fish Juvenile and adult sole samples were supplied by Seafresh Farms (Scotland) Ltd., Hunterston. O-Group juveniles weighed between 15-34 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 standard pelleted diets containing fish meal 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 examination of different parts of the

233

intestine, the alimentary canal of adult fish was divided into stomach, foregut, midgut, 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 h before being killed by a blow to the head followed by deep-freezing. After partial thawing, the intestine could be readily excised, weighed and homogenised with distilled water to give a 1:lO homogenate. Most analyses were carried out by using a further dilution of this sample to give a 1:40 homogenate. The lumen contents were included in all homogenates unless otherwise stated. Chemicals Unless otherwise stated all chemicals were supplied by Sigma Chemical Co. The buffers used were 0.1 M HCl (pH LO), glycine-HCl (LO-1.7), citratephosphate (1.7-7.8)) glycine-NaOH (pH 7.8-10.5). Enzyme assay Leucine aminopeptidase was measured by the method of Mitz and Schleuter (1958) in which the hydrolysis of the peptide bond of L-leucinamide was measured spectrophotometrically at 238 nm. Intestinal homogenate (1:40,0.1 ml) was activated in a mixture containing water (2.0 ml), 0.025 M manganese chloride (0.1 ml) and pH 8.3 buffer (0.1 ml). This mixture (0.1 ml) was then added to a 0.125 M solution of leucinamide (1 ml), 0.125 M magnesium chloride (0.1 ml), water (1.2 ml) and buffer (0.1 ml). The reagent blank consisted of 0.0625 M leucine (2.0 ml), 0.125 M magnesium chloride (0.1 ml), water (0.3 ml) and buffer (0.1 ml). The change in absorbance was measured with time and the results expressed as units min-I; the unit of enzyme activity being defined as the amount of enzyme hydrolysing 1 ,umole of substrate per min. Ion activition was studied by replacing 0.025 M manganese chloride (0.1 ml) with 0.025 M magnesium chloride or 0.025 M ethylenediamine tetraacetic (EDTA) . The activity was also measured with distilled water and omitting the 0.125 M magnesium chloride from the final digest. Inhibitor studies were carried out by preincubating a 1:40 intestinal homogenate with inhibitor for 30 min at room temperature (18” C ) before assaying for residual activity as described above. RESULTS

Using leucinamide as substrate, a very sharp optimum pH was obtained at pH 8.3 (Fig. 1) and under the assay conditions described in the Methods and Materials section, but varying the temperature, it was shown that the optimum temperature for this enzyme was 50” C (Fig. 2). In the absence of divalent

234

PH

Fig. 1. Effect of pH on the activity of intestinal homogenates of juvenile Dover sole towards Lleucinamide.

loo-

+- IS‘5

c

x

z 50. m c? 25 I J

O 20

30

40

50

60

Temperature("Cl

Fig. 2. Effect of temperature on the activity of intestinal towards L-leucinamide.

homogenates

of juvenile Dover sole

metal ions (EDTA present in enzyme digests) there was very little activity. On inclusion of magnesium ions the level increased by a factor of five and in the presence of manganese ions there was a 28-fold increase in the rate of hydrolysis of leucinamide (Fig. 3). Whole homogenates had about four times as much activity as the supernatant solutions from centrifuged homogenates (Fig. 4) and in an examination of homogenates from different sections of an adult gut (Fig. 5) the greatest proportion of activity was present in the midgut

235

Time (min) Fig.3.Effect of metal ions on the activity of intestinal

homogenates

of juvenile Dover sole towards

L-leucinamide.

Time Imin)

Fig. 4. Leucine aminopeptidase in centrifuged ( o-o ) , from juvenile Dover sole.

(o-o)

and uncentrifuged

homogenates

followed by the foregut, with only small amounts in the stomach, hindgut and rectum. The effect of inhibitors was studied by preincubatingthe enzyme with inhibitors. Table 1 indicates that whilst all inhibitors gave some degree of reduction in activity, N-tosyl-L-lysine chloromethyl ketone (TLCK) was the only one to remove the enzyme action completely.

Fig. 5. Relative activity of leucine aminopeptidase

in sections of adult Dover sole.

TABLE 1 Effect of inhibitors Inhibitor

PCMB Iodoacetate N-Bromosuccinimide N-Ethylmaleimide PMSF TLCK TPCK No inhibitor

on leucine aminopeptidaee

from Dover sole intestine

Activity U/g protein

Activity retained

4000 2260 1600 2000 1830 2800 0 5530

72 41 29 36 33 51 0 100

(%)

PCMB =p-chloromercuribenzoate; PMSF =phenyl methyl sulphonyl fluoride; TLCK = N-tosylL-lysine chloromethyl ketone; TPCK = N-tosyl-L-phenylalanine chloromethyl ketone. All inhibitors were used at a concentration of 10 mM in the preincubation mixture except for iodoacetate (30 mM) , TLCK (2 mM) and TPCK (3 mM) .

DISCUSSION

In the present study, leucine aminopeptidase has been assayed using leucinamide as substrate. This substrate has been used by most workers for biochemical investigation on this enzyme; however, for staining of tissues and gel electrophoreses L-leucylnaphthylamide has been employed (Alliot et al., 1977; Hirji and Courtney, 1982). The leucine aminopeptidase from Dover sole had a very sharp optimum at pH 8.3 (Fig. 1) . This compares with a value of pH 7.4 quoted for carp (Khablyuk and Proskuryakov, 1983a) and while the pH optimum may vary depending on the substrate used, most optima for mam-

237

malian leucine aminopeptidases occur in the region of pH 9 (Delange and Smith, 1971). The enzyme from swine kidney had a broad pH profile with maximum activity occurring between pH 7.8 and 9.0 (Himmelhoch, 1970). pH Values of ~7.0 were recorded in extracts of digestive tract from freshly killed Dover sole (unpublished results). While this suggests that gut pH is less than optimal for LAP activity, it is possible that ‘in viva’ pancreatic secretions may produce the necessary alkaline conditions in the distal regions of the gastrointestinal tract. Under the conditions used in the present study the Dover sole enzyme showed maximum activity at 50’ C ( Fig. 2 ) . This is in agreement with the temperature optima recorded for the hydrolysis of casein by alkaline proteases in Dover sole by Clark et al. ( 1986). In the present study the temperature optimum showed that LAP activity at temperatures less than 20°C was less than 10% of the optimum activity possible. Similar experiments with metabolic enzymes from other fish species have shown that although maximum activity often occurs at temperatures of 30-50’ C, i.e., greater than the environmental temperature, prolonged exposure to temperatures in excess of 50’ C usually result in thermal denaturation. When homogenates were centrifuged there was a four-fold loss of activity, suggesting that most of the enzyme was attached to the gut lining; however, the pH of optimum activity was the same for both centrifuged and uncentrifuged preparations. Dover sole does not have a well-defined gut structure; it has no pyloric caeca and the alimentary canal consists of a single tube penetrating into the body cavity (De Groot, 1971). Although the adult shows a bulging of the tube in the stomach area, the subdivision of the gut is somewhat arbitrary. Nevertheless, leucine aminopeptidase is located mainly in the midgut and foregut areas, with only small amounts in the stomach, hindgut and rectum (Fig. 5). Leucine aminopeptidase from bovine lens tissue has been shown to be activated by manganese and magnesium ions and to be inhibited by chelating agents and p-chloromercuribenzoate (Worthington Enzyme Manual, 1972 ) , and the enzyme from carp hepatopancreas (Khablyuk and Proskuryakov, 1983b) was also shown to be activated by the above metal ions. In the present work the effect of ions and the chelating agent, EDTA, is clearly demonstrated in Fig. 3 where there is five-fold enhancement of activity by inclusion of magnesium ions as compared with the absence of divalent metal ions in the presence of EDTA, and a 28-fold increase in activity in the presence of manganese ions. A number of inhibitors were tested at concentrations previously indicated in the literature. The most striking feature of the results (Table 1) is the complete inhibition by 3 mM TPCK. This substance has been shown by Schoellmann and Shaw (1963) to act by irreversible attachment by alkylating histidine residues and to give maximum inhibition at the pH of optimum activity of the enzyme. Other inhibitors used in the present study only showed less degrees of inhibition. These inhibitors were chosen for the variety of groups which they

attack: phenylmethylsulphonyl fluoride for serine residues (Turini et al., 1969)) N-bromosuccinimide for tryptophan (Spande and Witkop, 1967)) p-chloromercuribenzoate and N-ethylmaleimide for sulphydryl groups (Riordan and Vallee, 1967)) and iodoacetate by the addition of carboxymethyl groups to the side chains of amino acid residues such as lysine, methionine and cysteine (Gurd, 1967). It appears, therefore, that for leucine aminopeptidase from the intestine of Dover sole a histidine group which is susceptible to attack by TPCK is the most important feature of the active centre. In the studies of Overnell (1973 ) it was established that in addition to the pyloric caeca, leucine aminopeptidase was also present in the spleen and kidney of the cod. It was therefore concluded that in the cod this enzyme might have some function other than digestion. However, the concentrations of leutine aminopeptidase in the midgut and foregut regions of the alimentary canal suggest that this enzyme does have a digestive role in the Dover sole. ACKNOWLEDGEMENT

The authors wish to acknowledge ate) for the support of this work.

the SERC (Marine

Technology

Director-

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239 Khablyuk, V. V. and Proskuryakov, M. T., 1983b. Purification and some properties of leucine aminopeptidase from carp hepatopancreas. Prikl. Biokhim. Mikrobiol., 19: 427-430. Linderstrom-Lang, K., 1929. Uber Darmrepsin. Hoppe-Seyler’s Z. Physiol. Chem., 182: 151-174. Mitz, M. A. and Schleuter, R. J., 1958. Direct spectrophotometric measurement of the peptide bond: application to the determination of acylase, I. Biochim. Biophys. Acta, 27: 168. Overnell, J., 1973. Digestive enzymes of the pyloric caeca and of their associated mesentery in the cod (Gadus morhua) . Comp. Biochem. Physiol. B, 46: 519-531. Riordan, J. F. and Vallee, B. L., 1967. Reactions with N-ethylmaleimide and P-mercuribenzoate. In: C. H. W. Him (Editor), Methods in Enzymology, Vol. XI. Academic Press, London, pp. 541-548. Schoellmann, G. and Shaw, E., 1963. Direct evidence for the presence of histidine in the active center of chymotrypsin. Biochemistry, 2: 252-255. Spande, T. F. and Witkop, B., 1967. Determination of the tryptophan content of proteins with N-bromosuccinimide. In: C. H. W. Him (Editor), Methods in Enzymology, Vol. XI. Academic Press, London, pp. 498-506. Turini, P., Kurooka, S., Steer, M., Corbascio, A. N. and Singer, T. P., 1969. The action of phenylmethylsufonyl fluoride on human acetylcholinesterase, chymotrypsin and trypsin. J. Pharmacol. Exp. Ther., 167: 98-104. Worthington Enzyme Manual, 1972. Worthington Biochemical Corp., Freehold, NJ., pp. 115-116.