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The lipase and amylase of the common malayan toad Bufo melanostictus schneider
Comp. Biochem. Physiol.Vol. 105B, Nos 3/4, pp. 509-515, 1993
0305-0491/93 $6.00 + 0.00 © 1993 Pergamon Press Ltd
Printed in Great Britain
THE LIPASE A N D AMYLASE OF THE COMMON M A L A Y A N TOAD BUFO MELANOSTICTUS SCHNEIDER L. H. TEo,* T. W. Crn~N and L. L. TAN Department of Zoology, National University of Singapore, Kent Ridge Crescent, Singapore 0511
(Received 4 January 1993; accepted 12 February 1993) Abstract--l. The activities of lipase and amylase of Bufo melanostictus were studied to find out their responses to changes in pH and temperature (20-50°C). Their thermostability at 60°C was also determined. 2. The activities of the enzymes in each part of the digestive tract were determined for both the extracts of the tissues as well as the contents. 3. The effects of starvation on the activities of both enzymes were studied in both the normal room and an air-conditioned room.
INTRODUCTION
Bufo melanostictus is widely distributed in southeast Asia as well as India and southeastern China (Kampen, 1923; Smith, 1930). It is commonly found in rural, as well as urban areas, feeding on arthropods and molluscs with insects forming the main bulk of the diet (Berry and Bullock, 1962). Even plant materials have occasionally been found in their guts. It remains common in Singapore although urbanization has caused some frogs to become scarce. The pepsin and alkaline protease of this interesting species were recently studied by us (Teo et al., 1990) but information on other enzymes is completely unavailable. A search of literature shows that very little study has been carried out on lipase and amylase of amphibians, especially on the kinetic properties and quantitative distribution (Reeder, 1964; Vonk and Western, 1984). Toads and frogs are carnivorous and so, in the past, most of the work on their digestive enzymes had been concentrated on the proteases whilst lipases as well as carbohydrases were much neglected. Carnivorous animals also need lipids and carbohydrates to meet various requirements in their bodies and these two items are also found in their diets. This paper reports our studies on the lipase and amylase regarding their kinetic properties, quantitative distribution and the effect of starvation and temperature on their activities. MATERIALS AND METHODS
The toads were collected at night from areas around the university campus, kept in the laboratory and then immobilized by placing them in a cold room ( - 2 0 ° C ) . The pancreas and digestive tract were dissected by making transverse cuts across the *To whom correspondence should be addressed.
oesophagus and rectum. Ligatures were applied at various points along the tract to prevent contamination, so dividing the tract into the following parts: oesophagus, stomach, pancreas, small intestine and rectum. The small intestine was further differentiated into anterior, middle and posterior regions. Each portion was cut open and the contents flushed out with 1 ml ice-cold amphibian Ringer solution. These samples were centrifuged at 10,000g at 4°C for 15 min. The supernatants were retained and stored at - 2 0 ° C for no more than a week before assay. The rinsed gut tissues were placed in separate vials in an ice-bath. The mucosal layer of segment was separated from the muscle coat by scraping. Each tissue was blotted dry, weighed, and ice-cold Ringer's solution added (100 mg tissue to 1 ml solution). The portions were homogenized in a pre-chilled homogenizer. The extracts were centrifuged and the supernatants stored at - 2 0 ° C . A modified method of Myrtle and Zell (1975) used by Teo and Woodring (1985) was adopted for lipase assay. An aliquot of 0.2 ml triolein emulsion was mixed with 0.2 ml enzyme extract and 0.2 ml buffer. The reaction mixture was incubated for 10 rain at 37°C and the reaction stopped with the addition of 7.0 ml of chloroform-heptane (3:2) solution. The test-tubes were shaken vigorously with a vortex mixer and then centrifuged for 10 min. The upper layer was removed and 5 ml of the chloroform-heptane layer was pipetted into another tube with 10 ml saturated sodium bromide solution and 3.5 ml copper reagent. The tubes were vigorously shaken with a vortex mixer and centrifuged for 10 min. An aliquot of 3 ml of the top layer was transferred to a quartz cuvette containing 0.5 ml sodium diethyldithiocarbamate reagent. The absorbance was measured at 435 nm. The assay for amylase was carried out with 1% boiled starch or 1% glycogen solution. The reaction mixture consisted of I ml substrate solution, 0.3 ml
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enzyme extract and 0.7 ml buffer solution. The reaction mixture was incubated at 37°C for 1 hr in the presence of 2 drops of toluene. The maltose produced was determined with dinitrosalicylate reagent (Bernfeld, 1955). The absorbance was read at 530nm against a control in which the enzyme extract had been boiled for 3 rain. This control serves to offset any reducing sugar which might be present in the enzyme extract. To study the effect of high temperature on the stability of the enzymes, the freshly prepared enzyme extract was separated into several portions and incubated at 60°C for periods up to 160 min and then quickly chilled in ice-cold water before storage at - 2 0 ° C for 1-3 days. A portion not exposed to high temperature was used as the control. It was immediately stored at - 2 0 ° C for the same period of time and its activity assumed to be 100%. Enzyme activity was determined in the manner described above. To study the effect of starvation, toads were randomly divided into two groups after collection. One group was immediately dissected for enzyme assays; the second group was starved for 5 days at room temperature before use in enzyme assays. They were given a constant supply of water. To examine the interaction between temperature and starvation 80 toads were divided randomly into four groups. Two were maintained at room temperature (28 + 3°C) and the remaining two at 24 + 3°C. In each case one group was provided with water only and the other group was force-fed Tenebrio molitor beetles on alternate days. After 2 weeks all toads were sacrificed.
RESULTS
Lipase showed maximum activity at pH 8.0 (Fig. 1) and its activity decreased rapidly outside the narrow optimum range of pH 8.0 _+ 0.3 but the highest pH along the digestive tract of this toad was only pH 7.6
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pH Fig. 1. The pH-activity curve of lipase of the Malayan common toad.
TEMPERATURE
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Fig. 3. The temperature-activity curve of lipase of the Malayan common toad.
511
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presented were in terms of total units in each ml of enzyme extract and total units in each part of the digestive tract. The former indicates the quantity present in each ml of enzyme extract and so is an estimation of the concentration of lipase present in each unit volume of the extract. The latter gives an estimation of the contribution of lipase from each part of the gut and so allows us to compare the contribution of lipase by different parts of the gut. From the tissue extracts, the highest concentration of lipase was found in the pancreas which was followed by the stomach, oesophagus and the middle small intestine, in that order. The contents of the rectum and the middle small intestine had the highest concentrations of lipase. Due to its size, the mucosa of the stomach had the largest amount of lipase. This even exceeded the total units from other parts of the digestive tract added together. However, the lipase activity of the gastric contents was only significantly higher than that of the anterior small intestine. On the other hand, the contents of the oesophagus contained slightly more lipase than the contents of other parts of the digestive tract. The anterior small intestine not only had the lowest concentration of
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Fig. 6. The Arrhenius plot of the effect of temperature on the activity of amylase.
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optimum activity also occurred at 40°C under the present conditions of experimentation. The average Q]0 value for the temperature of 20-40°C was only 1.10 with slight differences at the various temperature ranges. The Arrhenius plot yielded a much lower value for the energy of activation (EA = 2000 cal/mol) than of lipase (Fig. 6). Lipase lost its activity completely even after 4 min of treatment at 60°C. The response of amylase to high-temperature treatment is shown in Fig. 6. A portion of lipase was not stable at 60°C and lost 59% activity after only 6 rain of treatment but the rest of it seemed to be very resistant to high temperature treatment so that treatment from 10 to 160 rain (not shown in Fig. 6) only resulted in 63-65% inactivation. The probit-log plot (Fig. 7) gives an estimate of 4.5 min for 50% inactivation of amylase at 60°C. Table 1 shows the quantitative distribution of lipase in different parts of the digestive tract. The data
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TEMPERATURE
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Fig. 5. The temperature-activity curve of amylase of the Malayan common toad.
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60
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TREATMENT
TIME
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Fig. 7. Inactivation of amylase by high temperature (60°C) treatment for various periods.
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512
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the activity of amylase. lipase, also had the smallest quantity of total lipase in its contents. The stomach had the lowest C/T ratio and this may indicate that very little lipase is secreted by the gastric mucosa into the gastric lumen. The highest C/T ratio was that of the rectum and this was most likely due to input from the small intestine. The amylase was assayed with two different substrates. Table 2 shows that the activity on glycogen by extracts from the mucosa of all parts of the digestive tract, except the pancreas, was lower than that on starch. In terms of units of activity in each ml of the enzyme extract, the activity of pancreatic extracts on glycogen was 235% of that on starch. However, the activity of mucosal extract of the stomach on starch was 13.6 times that on glycogen. The mucosal extracts of other parts of the gut also showed higher activities on starch than on glycogen by factors ranging from slightly above six to slightly below 10, but higher activities on glycogen than on starch were found in the contents. The extracts from the contents of the anterior small intestine had more than three times the activity on glycogen than on starch but fewer differences were found with the extracts from the contents of the stomach and the middle small intestine. It is obvious from Table 3 that the specific activity of lipase of pancreatic extract was about the same as
that of the mucosal extracts of both the oesophagus and stomach and that the posterior small intestine had slightly higher specific activity than the anterior small intestine. The highest specific activity of the contents was recorded in both the oesophagus and stomach. With the exception of the anterior small intestine, the contents of all parts studied showed only small differences in specific activity of lipase. It is also interesting that, in terms of units of lipase per ml of enzyme extract, the pancreatic extract had a much higher value than the mucosal extract of the oesophagus but their specific activities showed no significant differences. The tissue extracts of all parts of the gut, except the pancreas had higher specific activities on starch whilst that of the pancreatic tissues had higher specific activity on glycogen (Table 4). The differences in specific activities on glycogen between extracts of pancreatic tissues and extracts of tissues of other parts of the gut on glycogen were much larger than the differences between them on starch. In the contents of various parts of the gut, there were higher specific activities on glycogen than on starch. The biggest difference was found in the anterior small intestine and the smallest difference was found in the middle small intestine. The anterior small intestine had both the highest specific activity on glycogen and starch as well as the highest U/mi enzyme of amylase in its contents. Toads kept at a higher temperature (in normal room) for 14 days had slightly higher activities of amylase than those kept in an air-conditioned room. In the case of lipase, the difference was even greater (29%) although the difference in temperature was only 4°C. In toads which had been starved for 14 days, there was hardly any difference in the activity of amylase between toads kept in the normal room and those kept in the air-conditioned room but the difference in lipase activity was by a factor of 3.8. In toads starved for 5 days, the activities of both amylase and lipase in the pancreas were not affected by starvation. The activities of amylase in the intestine were also hardly affected by starvation but the activity of lipase of the starved toads was only about half that in fed toads. Thus the longer the period of starvation, the bigger was its effect on the activity of intestinal lipase.
Table 1. The distribution of lipase [mean (SE)] in the digestive system of Bufo melanosticlus U/ml enzyme extract Source Oesophagus Stomach Intestine 1 Intestine 2 Intestine 3 Rectum Pancreas
Total U/organ
Tissue
Contents
Tissues
Contents
C/T
15.32 (0.78) 17.27 (1.79) 7.07 (0.28) 12.02 (1.02) 7.73 (0.89) 0.90 (0.06) 21.32 (0.89)
7.63 (0.18) 7.15 (0.36) 5.13 (0.38) 12.15 (0.90) 7.67 (0.72) 14.14(1.56)
13.79 (0.69) 61.31(6.35) 7.78 (0.62) 7.57 (0.49) 3.48 (0.40) 0.63 (0.04) 9.59 (0.40)
16.48 (0.39) 14.51 (0.72) 6.21 (0.50) 13.37(1.03) 12.58 (1.18) 12.30(0.69)
1.20 0.24 0.80 1.77 3.61 19.52
! U: gM oleic acid min -I ml -I or organ. C/T: total U in contents/total U in tissues. Intestine 1-3: anterior, middle and posterior small intestine.
T o a d amylase and lipase
513
Table 2. The distribution of amylase [mean (SE)] in the digestive system of Bufo melanostictus U/ml enzyme extract Source
Tissue
Total U/organ
Contents
Tissues
Contents
C/l"
Substrate: glycogen Oesophagus 6.95 (0.74) Stomach 2.38 (0.27) Intestine 1 7.67 (1.76) Intestine 2 6.16 (1.62) Intestine 3 7.04 (1.61) Rectum 9.24 (2.54) Pancreas 336.31 (31.53)
8.46 (!.08) 5.76 (I.76) 33.31 (1.35) 9.43 (1.83) 15.02 0.79) 16.17 (2.45)
6.26 (0.67) 8.45 (0.96) 8.48 (1.94) 3.88 (1.02) 3.17 (0.72) 6.47 (I .78) 151.47 (14.19)
18.27 (2.33) 11.69 (3.57) 40.31 (1.63) 10.37 (2.01) 24.63 (2.93) 14.07 (2.13)
2.92 1.38 4.75 2.67 7.77 2.17
Substrate: starch Oesophagus Stomach Intestine l Intestine 2 Intestine 3 Rectum Pancreas
4.88(0.62) 3.40 (0.27) 10.43 (0.56) 7.36 (0.50) 7.09 (0.54) 9.49 (0.60)
60.61 (4.59) I 15.39 (4.51) 54.33 (3.32) 26.82 (1.59) 22.72 (2.02) 43.47 (4.41) 64.69 (4.18)
10.54 (1.34) 6.90 (0.54) 12.62 (0.68) 8.09 (0.55) 11.63 (0.88) 8.26 (0.52)
0.17 0.06 0.23 0.30 0.51 0.19
67.19 (5.10) 32.49 (1.27) 49.14 (3.00) 42.58 (2.60) 50.45 (4.50) 62.08 (6.30) 143.75 (9.30)
1 U: #M maltose hr -~ ml -~ or per organ. C/T: Total U in contents/total U in tissues. Intestine 1-3: anterior, middle and posterior small intestine.
DISCUSSION
The optimal pH of lipase of Bufo melanostictus is the same as that of Rana esculenta (Scapin and Lambert-Gardini, 1979) except that the optimal pH range is slightly narrower in the former species. This is lower than the optimal pH (8.2-9.2) of mammalian lipase (Vonk and Western, 1984). The optimal pH of amylase is also the same as the amylase of Rana esculenta (Scapin and Lambert-Gardini, 1979) and Rana castesbiana (McGeachin and Welbourne, 1971) and slightly higher than that of mammal (optimal pH 6.9) (Vonk and Western, 1984). This optimum pH makes the amylase of this toad well adapted to the pH conditions in various parts of the intestine. The lipase of Bufo melanostictus responded moderately to the increase of temperature and its estimated activation energy was slightly on the low side. The amylase showed very little response to the increase of temperature and its activation energy was very low. In contrast, the pepsin and alkaline protease of this toad showed a better response to the increase of temperature (Teo et al., 1990). The amylase, and in particular, the lipase of Bufo melanostictus were both very unstable at 60°C. It was also previously reported that the amylase and lipase of Rana esculenta were unstable at 50°C (Scapin and Lambert-Gardini, 1979). In both species, lipase was less stable than amylase. The amylase was similar to the alkaline protease of the same toad in having a Table 3. The specific activities ~ moi oleic acid hr - l m g - ' protein- z) of lipase in the tissues and lumen of various parts of the digestive tract of Bufo melanostictus Region of gut Oesophagus Stomach Anterior small intestine Middle small intestine Posterior small intestine Rectum Pancreas
Tissue
Contents
448.5 + 17.5 423.0 _+43.8 188.5 + 7.6 369.9 + 13.3 265.1 + 15.1 28.8 +_ 1.8 423.6 + 17.6
482.1 _+ 10.0 457.4 + 15.8 152.5 + 11.3 409.5 + 10.1 430.0 + 20.0 394.5 + 14.9
portion of it very stable at high temperature (Teo et al., 1990). It is thought that there are perhaps two components of both the alkaline protease and amylase, one of them being highly thermostable. A similar observation was made in the sucrase of Drosophila melanogaster (Marzluf, 1969) and the amylase of the green mussel (Sahapathy and Teo, 1992). In the latter, the 50% inactivation time at 50°C was only 4.4 min and so it is more thermolabile than the amylase of Bufo melanostictus. It was quite surprising that the gastric mucosal extract had such a high total lipase activity although much of the lipase in the mucosa was proabably not secreted into the gastric lumen. Although the oesophageal mucosa did not have such a high total lipase activity, it seemed that much of it was secreted into its lumen, as judged by the relatively high activity level in its contents. The latter was one of the highest activity levels recorded. However, the contents of the oesophagus probably do not stay long in it but pass quickly, together with food, into the stomach where the acidic conditions would not allow lipase to be active. The lipase present in the gastric lumen might become denatured in the acidic conditions before it reached the anterior small intestine. That might explain why the latter had the lowest level of lipase activity in its lumen. Based on this argument, the digestion of fat seems to depend a lot on lipase supplied by the pancreas and the small intestine. The rectal mucosa had a low level of lipase activity but its contents had lipase activity comparable to that of the middle and posterior small intestine and so some of the lipase present in its lumen probably had its origin in the more anterior part of the intestine, as well as the pancreas. The contents of the anterior small intestine had the lowest total units of lipase, the lowest units of lipase per ml of contents and also the lowest specific activity of lipase. The anterior small intestine is least important in the digestion of lipid. The important regions in the digestion of lipid seemed
L. H. TEo et al.
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Table 4. The specificactivities (mg maltose hr-m mg protein-m) of amylaseon starch and glycogen in different parts of the gut of Bufo melanostictus Stash Region of gut Oesophagus Stomach Intestine 1 Intestine 2 Intestine 3 Rectum Pancreas
~yco~n
Tissue
Contents
Tissue
Contents
11.80 __ 0.09 4.86 + 0.19 7.88 4- 0.24 7.894-0.26 10.39 4- 0.93 11.964-0.20 17.15 4- l . l l
1.85 4- 0.09 1.02 4- 0.05 1.86 + 0.10 1.49 4- 0.05 2.39 + 0.09 1.594-0.05
1.22 4- 0.13 0.35 4- 0.04 1.23 4- 0.28 1.14 __ 0.30 1.45 4- 0.33 1.784-0.49 40.16 __ 3.76
3.21 _+0.41 1.73 __ 0.53 5.94 4- 0.24 1.91 4-0.37 5.06 4- 0.60 2.71 4-0.41
Intestine 1: anterior small intestine. Intestine 2: middle small intestine. Intestine 3: posterior small intestine.
to be the middle and posterior regions of the small intestine. The contents of rectum also had high lipase activity but as it is the last region of the intestine, the food probably does not remain in it long enough for sufficient absorption of products of digestion to take place across its walls. Although the oesophagus had high activities of lipase and moderate activity of amylase in its contents, it probably has a small role in digestion as foods normally pass through it directly into the stomach. Although the pancreatic extract had one of the highest specific activities on lipid, and its total unit of lipase per ml of enzyme was also the highest, the total of units of lipase in the whole pancreas was lower than that of stomach and oesophagus and also not very much higher than that of the mucosal extracts of the anterior and middle small intestine. Its total of units was only half of that present in the entire small intestine. Thus it may be concluded that the contribution of the pancreas to lipid digestion was not the most important one. The anterior small intestine, on the other hand, had the highest activity on glycogen in terms of the total units per organ and total units per ml of enzyme extract and specific activity. All these indicate that the anterior small intestine is most important in the digestion of glycogen. The posterior small intestine is probably only second to the anterior small intestine in its importance to the digestion of glycogen. The higher activities on glycogen than on starch found in the small intestine and rectum might be explained by the input of amylase from the pancreas. The lumen
of rectum would also receive input from the small intestine. But the activities of extracts from the contents of the oesophagus and stomach were also higher on glycogen than on starch. This either indicates regurgitation of enzymes from the anterior small intestine to the stomach, and even oesophagus, or the amylase isozyme which showed higher preference for glycogen as a substrate was secreted in larger proportions (out of its proportion present in mucosa of these two regions) in response to the diets of the toads. Pancreas is the most important source of amylase, with glycogen as the substrate. Its total activities far exceed those of all other parts of the gut added together and its specific activity, as well as activities per ml of enzyme extract, far exceed those of any other parts of the digestive tract. The contribution of the pancreas to the digestion of starch is far below its contribution to the digestion of glycogen. With regards to the activities on starch and lipid, the pancreatic extracts maintained the same levels of activities even after 5 days of starvation. The amylase activity of the small intestine was only slightly affected by starvation after 14 days. There was only a slight difference in its activity at the two different temperatures. On the other hand, lipase activity was much affected by both starvation and temperature. It had a greater dependence on these two factors than proteases (Teo et aL, 1990). In Rana esculenta, pancreatic lipase was not affected by starvation but pancreatic amylase was much affected, as its activity fell by 39% (Scapin and Lambert-Gardini 1979). In
Table 5. Effect of starvation on the enzymic activities [mean (SE)] in Bufo raelanostictus Normal room Enzyme (source) Starved for 5 days Amylase (Pan) Amylase (Int) Lipase (Pan) Lipase (Int) Starved for 14 days Amylase (Int) Lipase (Int)
Fed
11.97 (0.91) 72.8 (8.3)
Amylase:/tM maltose hr -I. Lipase:/~M oleic acid m i n - k Pan: pancreas; lnt: small intestine. Normal room: 28 + 3°C. Air-conditioned room: 24 + 3°C.
Starved
9.24 (0.64) 49.1 (4.1)
Air-conditioned room Fed
Starved
11.96 (0.15) 9.74 (0.72) 147.8 (12.0) 68.25 (3.8)
11.93 (0.12) 8.50 (0.44) 141.0 (8.8) 34.50 (1.90)
9.52 (0.68) 51.8 (6.8)
8.82 (0.68) 12.8 (1.9)
Toad amylase and lipase the bull frog, pancreatic amylase fell by 40% and intestinal amylase decreased by 70% after animals had been starved for 1 week; in the leopard frog, the respective figures were 37 and 60% (McGeachin and Welbourne 1971). It is interesting that keeping toads at two different temperatures, with only a small difference of 4°C, resulted in differences in the activities of both amylase and lipase in the intestine. The difference was even more spectacular in the case of lipase which showed a decrease of 29% activity in the fed toads and 74% in the starved toads at the lower temperature. It may be interesting to point out that the amylase activity is not only less affected by starvation but is also less affected by environmental temperature, since the decrease in activity was less drastic when toads were kept at a lower temperature. It is not certain whether this has anything to do with the fact that toads may depend heavily on glycogen as a source of energy for general motor activities. It would be an advantage to the toads to have a good supply of amylase at any time so that when food is available, the enzyme is there in sufficient quantities to digest it in order that the source of energy for muscles be readily available. REFERENCES
Bernfeid P. (1955) Amylases, ct and ft. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 149-158. Academic Press, New York.
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Berry P. Y. and Bullock J. A. (1962) The food of the common Malayan toad, Bufo melanostictus Schneider. Copeia 1962, 736-741. Kampen P. N. van (1923) The Amphibia of the IndoAustralian Archipelago. E. J. Brill, London. Marzluf G. A. (1969) Studies of trehalase and sucrase of Drosophila melanogaster. Archs Bioehem. Biophys. 134, 8-18. McGeachin R. L. and Welbourne W. P. (1971) Amylase in tissues of the bullfrog, Rana catesbeiana and the leopard frog, Rana pipiens. Comp. Biochem. Physiol. 38A, 457-460. Myrtle J. F. and Zell W. J. (1975) Simplified photometric copper-soap method for rapid assay of serum lipase activity. Clin. Chem. 21, 1469-1473. Reeder W. G. (1964) The digestive system. In Physiology oJ Amphibia (Edited by Moore J. A.), pp. 99-149. Academic Press, London. Sabapathy U. and Teo L. H. (1992) A kinetic study of the or-amylase from the digestive gland of Perna viridis L. Comp. Biochem. Physiol. 101B, 73-77. Scapin S. and Lambert-Gardini S. (1979) Digestiveenzymes in the exocrine pancreas of the frog Rana esculenta. Comp. Biochem. Physiol. 62A, 691~597. Smith M. A. (1930) The Reptilia and Amphibia of the Malay Peninsula. Bull. Raffles Mus. Singapore 3, 1-149. Teo L. H., Chen T. W. and Tan L. L. (1990) The proteases of the common Malayan toad Bufo melanostictus Schneider. Comp. Biochem. Physiol. 96II, 715-720. Teo L. H. and Woodring J. P. (1985) Digestive enzymes in the house cricket Acheta domesticus with special reference to amylase. Comp. Biochem. Physiol. 82A, 871-877. Vonk H. J. and Western J. R. H. (1984) Comparative Biochemistry and Physiology of Enzymatic Digestion. Academic Press, London.
0305-0491/93 $6.00 + 0.00 © 1993 Pergamon Press Ltd
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THE LIPASE A N D AMYLASE OF THE COMMON M A L A Y A N TOAD BUFO MELANOSTICTUS SCHNEIDER L. H. TEo,* T. W. Crn~N and L. L. TAN Department of Zoology, National University of Singapore, Kent Ridge Crescent, Singapore 0511
(Received 4 January 1993; accepted 12 February 1993) Abstract--l. The activities of lipase and amylase of Bufo melanostictus were studied to find out their responses to changes in pH and temperature (20-50°C). Their thermostability at 60°C was also determined. 2. The activities of the enzymes in each part of the digestive tract were determined for both the extracts of the tissues as well as the contents. 3. The effects of starvation on the activities of both enzymes were studied in both the normal room and an air-conditioned room.
INTRODUCTION
Bufo melanostictus is widely distributed in southeast Asia as well as India and southeastern China (Kampen, 1923; Smith, 1930). It is commonly found in rural, as well as urban areas, feeding on arthropods and molluscs with insects forming the main bulk of the diet (Berry and Bullock, 1962). Even plant materials have occasionally been found in their guts. It remains common in Singapore although urbanization has caused some frogs to become scarce. The pepsin and alkaline protease of this interesting species were recently studied by us (Teo et al., 1990) but information on other enzymes is completely unavailable. A search of literature shows that very little study has been carried out on lipase and amylase of amphibians, especially on the kinetic properties and quantitative distribution (Reeder, 1964; Vonk and Western, 1984). Toads and frogs are carnivorous and so, in the past, most of the work on their digestive enzymes had been concentrated on the proteases whilst lipases as well as carbohydrases were much neglected. Carnivorous animals also need lipids and carbohydrates to meet various requirements in their bodies and these two items are also found in their diets. This paper reports our studies on the lipase and amylase regarding their kinetic properties, quantitative distribution and the effect of starvation and temperature on their activities. MATERIALS AND METHODS
The toads were collected at night from areas around the university campus, kept in the laboratory and then immobilized by placing them in a cold room ( - 2 0 ° C ) . The pancreas and digestive tract were dissected by making transverse cuts across the *To whom correspondence should be addressed.
oesophagus and rectum. Ligatures were applied at various points along the tract to prevent contamination, so dividing the tract into the following parts: oesophagus, stomach, pancreas, small intestine and rectum. The small intestine was further differentiated into anterior, middle and posterior regions. Each portion was cut open and the contents flushed out with 1 ml ice-cold amphibian Ringer solution. These samples were centrifuged at 10,000g at 4°C for 15 min. The supernatants were retained and stored at - 2 0 ° C for no more than a week before assay. The rinsed gut tissues were placed in separate vials in an ice-bath. The mucosal layer of segment was separated from the muscle coat by scraping. Each tissue was blotted dry, weighed, and ice-cold Ringer's solution added (100 mg tissue to 1 ml solution). The portions were homogenized in a pre-chilled homogenizer. The extracts were centrifuged and the supernatants stored at - 2 0 ° C . A modified method of Myrtle and Zell (1975) used by Teo and Woodring (1985) was adopted for lipase assay. An aliquot of 0.2 ml triolein emulsion was mixed with 0.2 ml enzyme extract and 0.2 ml buffer. The reaction mixture was incubated for 10 rain at 37°C and the reaction stopped with the addition of 7.0 ml of chloroform-heptane (3:2) solution. The test-tubes were shaken vigorously with a vortex mixer and then centrifuged for 10 min. The upper layer was removed and 5 ml of the chloroform-heptane layer was pipetted into another tube with 10 ml saturated sodium bromide solution and 3.5 ml copper reagent. The tubes were vigorously shaken with a vortex mixer and centrifuged for 10 min. An aliquot of 3 ml of the top layer was transferred to a quartz cuvette containing 0.5 ml sodium diethyldithiocarbamate reagent. The absorbance was measured at 435 nm. The assay for amylase was carried out with 1% boiled starch or 1% glycogen solution. The reaction mixture consisted of I ml substrate solution, 0.3 ml
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enzyme extract and 0.7 ml buffer solution. The reaction mixture was incubated at 37°C for 1 hr in the presence of 2 drops of toluene. The maltose produced was determined with dinitrosalicylate reagent (Bernfeld, 1955). The absorbance was read at 530nm against a control in which the enzyme extract had been boiled for 3 rain. This control serves to offset any reducing sugar which might be present in the enzyme extract. To study the effect of high temperature on the stability of the enzymes, the freshly prepared enzyme extract was separated into several portions and incubated at 60°C for periods up to 160 min and then quickly chilled in ice-cold water before storage at - 2 0 ° C for 1-3 days. A portion not exposed to high temperature was used as the control. It was immediately stored at - 2 0 ° C for the same period of time and its activity assumed to be 100%. Enzyme activity was determined in the manner described above. To study the effect of starvation, toads were randomly divided into two groups after collection. One group was immediately dissected for enzyme assays; the second group was starved for 5 days at room temperature before use in enzyme assays. They were given a constant supply of water. To examine the interaction between temperature and starvation 80 toads were divided randomly into four groups. Two were maintained at room temperature (28 + 3°C) and the remaining two at 24 + 3°C. In each case one group was provided with water only and the other group was force-fed Tenebrio molitor beetles on alternate days. After 2 weeks all toads were sacrificed.
RESULTS
Lipase showed maximum activity at pH 8.0 (Fig. 1) and its activity decreased rapidly outside the narrow optimum range of pH 8.0 _+ 0.3 but the highest pH along the digestive tract of this toad was only pH 7.6
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pH Fig. 1. The pH-activity curve of lipase of the Malayan common toad.
TEMPERATURE
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Fig. 3. The temperature-activity curve of lipase of the Malayan common toad.
511
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presented were in terms of total units in each ml of enzyme extract and total units in each part of the digestive tract. The former indicates the quantity present in each ml of enzyme extract and so is an estimation of the concentration of lipase present in each unit volume of the extract. The latter gives an estimation of the contribution of lipase from each part of the gut and so allows us to compare the contribution of lipase by different parts of the gut. From the tissue extracts, the highest concentration of lipase was found in the pancreas which was followed by the stomach, oesophagus and the middle small intestine, in that order. The contents of the rectum and the middle small intestine had the highest concentrations of lipase. Due to its size, the mucosa of the stomach had the largest amount of lipase. This even exceeded the total units from other parts of the digestive tract added together. However, the lipase activity of the gastric contents was only significantly higher than that of the anterior small intestine. On the other hand, the contents of the oesophagus contained slightly more lipase than the contents of other parts of the digestive tract. The anterior small intestine not only had the lowest concentration of
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Fig. 6. The Arrhenius plot of the effect of temperature on the activity of amylase.
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optimum activity also occurred at 40°C under the present conditions of experimentation. The average Q]0 value for the temperature of 20-40°C was only 1.10 with slight differences at the various temperature ranges. The Arrhenius plot yielded a much lower value for the energy of activation (EA = 2000 cal/mol) than of lipase (Fig. 6). Lipase lost its activity completely even after 4 min of treatment at 60°C. The response of amylase to high-temperature treatment is shown in Fig. 6. A portion of lipase was not stable at 60°C and lost 59% activity after only 6 rain of treatment but the rest of it seemed to be very resistant to high temperature treatment so that treatment from 10 to 160 rain (not shown in Fig. 6) only resulted in 63-65% inactivation. The probit-log plot (Fig. 7) gives an estimate of 4.5 min for 50% inactivation of amylase at 60°C. Table 1 shows the quantitative distribution of lipase in different parts of the digestive tract. The data
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TEMPERATURE
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Fig. 5. The temperature-activity curve of amylase of the Malayan common toad.
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60
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TREATMENT
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Fig. 7. Inactivation of amylase by high temperature (60°C) treatment for various periods.
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512
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the activity of amylase. lipase, also had the smallest quantity of total lipase in its contents. The stomach had the lowest C/T ratio and this may indicate that very little lipase is secreted by the gastric mucosa into the gastric lumen. The highest C/T ratio was that of the rectum and this was most likely due to input from the small intestine. The amylase was assayed with two different substrates. Table 2 shows that the activity on glycogen by extracts from the mucosa of all parts of the digestive tract, except the pancreas, was lower than that on starch. In terms of units of activity in each ml of the enzyme extract, the activity of pancreatic extracts on glycogen was 235% of that on starch. However, the activity of mucosal extract of the stomach on starch was 13.6 times that on glycogen. The mucosal extracts of other parts of the gut also showed higher activities on starch than on glycogen by factors ranging from slightly above six to slightly below 10, but higher activities on glycogen than on starch were found in the contents. The extracts from the contents of the anterior small intestine had more than three times the activity on glycogen than on starch but fewer differences were found with the extracts from the contents of the stomach and the middle small intestine. It is obvious from Table 3 that the specific activity of lipase of pancreatic extract was about the same as
that of the mucosal extracts of both the oesophagus and stomach and that the posterior small intestine had slightly higher specific activity than the anterior small intestine. The highest specific activity of the contents was recorded in both the oesophagus and stomach. With the exception of the anterior small intestine, the contents of all parts studied showed only small differences in specific activity of lipase. It is also interesting that, in terms of units of lipase per ml of enzyme extract, the pancreatic extract had a much higher value than the mucosal extract of the oesophagus but their specific activities showed no significant differences. The tissue extracts of all parts of the gut, except the pancreas had higher specific activities on starch whilst that of the pancreatic tissues had higher specific activity on glycogen (Table 4). The differences in specific activities on glycogen between extracts of pancreatic tissues and extracts of tissues of other parts of the gut on glycogen were much larger than the differences between them on starch. In the contents of various parts of the gut, there were higher specific activities on glycogen than on starch. The biggest difference was found in the anterior small intestine and the smallest difference was found in the middle small intestine. The anterior small intestine had both the highest specific activity on glycogen and starch as well as the highest U/mi enzyme of amylase in its contents. Toads kept at a higher temperature (in normal room) for 14 days had slightly higher activities of amylase than those kept in an air-conditioned room. In the case of lipase, the difference was even greater (29%) although the difference in temperature was only 4°C. In toads which had been starved for 14 days, there was hardly any difference in the activity of amylase between toads kept in the normal room and those kept in the air-conditioned room but the difference in lipase activity was by a factor of 3.8. In toads starved for 5 days, the activities of both amylase and lipase in the pancreas were not affected by starvation. The activities of amylase in the intestine were also hardly affected by starvation but the activity of lipase of the starved toads was only about half that in fed toads. Thus the longer the period of starvation, the bigger was its effect on the activity of intestinal lipase.
Table 1. The distribution of lipase [mean (SE)] in the digestive system of Bufo melanosticlus U/ml enzyme extract Source Oesophagus Stomach Intestine 1 Intestine 2 Intestine 3 Rectum Pancreas
Total U/organ
Tissue
Contents
Tissues
Contents
C/T
15.32 (0.78) 17.27 (1.79) 7.07 (0.28) 12.02 (1.02) 7.73 (0.89) 0.90 (0.06) 21.32 (0.89)
7.63 (0.18) 7.15 (0.36) 5.13 (0.38) 12.15 (0.90) 7.67 (0.72) 14.14(1.56)
13.79 (0.69) 61.31(6.35) 7.78 (0.62) 7.57 (0.49) 3.48 (0.40) 0.63 (0.04) 9.59 (0.40)
16.48 (0.39) 14.51 (0.72) 6.21 (0.50) 13.37(1.03) 12.58 (1.18) 12.30(0.69)
1.20 0.24 0.80 1.77 3.61 19.52
! U: gM oleic acid min -I ml -I or organ. C/T: total U in contents/total U in tissues. Intestine 1-3: anterior, middle and posterior small intestine.
T o a d amylase and lipase
513
Table 2. The distribution of amylase [mean (SE)] in the digestive system of Bufo melanostictus U/ml enzyme extract Source
Tissue
Total U/organ
Contents
Tissues
Contents
C/l"
Substrate: glycogen Oesophagus 6.95 (0.74) Stomach 2.38 (0.27) Intestine 1 7.67 (1.76) Intestine 2 6.16 (1.62) Intestine 3 7.04 (1.61) Rectum 9.24 (2.54) Pancreas 336.31 (31.53)
8.46 (!.08) 5.76 (I.76) 33.31 (1.35) 9.43 (1.83) 15.02 0.79) 16.17 (2.45)
6.26 (0.67) 8.45 (0.96) 8.48 (1.94) 3.88 (1.02) 3.17 (0.72) 6.47 (I .78) 151.47 (14.19)
18.27 (2.33) 11.69 (3.57) 40.31 (1.63) 10.37 (2.01) 24.63 (2.93) 14.07 (2.13)
2.92 1.38 4.75 2.67 7.77 2.17
Substrate: starch Oesophagus Stomach Intestine l Intestine 2 Intestine 3 Rectum Pancreas
4.88(0.62) 3.40 (0.27) 10.43 (0.56) 7.36 (0.50) 7.09 (0.54) 9.49 (0.60)
60.61 (4.59) I 15.39 (4.51) 54.33 (3.32) 26.82 (1.59) 22.72 (2.02) 43.47 (4.41) 64.69 (4.18)
10.54 (1.34) 6.90 (0.54) 12.62 (0.68) 8.09 (0.55) 11.63 (0.88) 8.26 (0.52)
0.17 0.06 0.23 0.30 0.51 0.19
67.19 (5.10) 32.49 (1.27) 49.14 (3.00) 42.58 (2.60) 50.45 (4.50) 62.08 (6.30) 143.75 (9.30)
1 U: #M maltose hr -~ ml -~ or per organ. C/T: Total U in contents/total U in tissues. Intestine 1-3: anterior, middle and posterior small intestine.
DISCUSSION
The optimal pH of lipase of Bufo melanostictus is the same as that of Rana esculenta (Scapin and Lambert-Gardini, 1979) except that the optimal pH range is slightly narrower in the former species. This is lower than the optimal pH (8.2-9.2) of mammalian lipase (Vonk and Western, 1984). The optimal pH of amylase is also the same as the amylase of Rana esculenta (Scapin and Lambert-Gardini, 1979) and Rana castesbiana (McGeachin and Welbourne, 1971) and slightly higher than that of mammal (optimal pH 6.9) (Vonk and Western, 1984). This optimum pH makes the amylase of this toad well adapted to the pH conditions in various parts of the intestine. The lipase of Bufo melanostictus responded moderately to the increase of temperature and its estimated activation energy was slightly on the low side. The amylase showed very little response to the increase of temperature and its activation energy was very low. In contrast, the pepsin and alkaline protease of this toad showed a better response to the increase of temperature (Teo et al., 1990). The amylase, and in particular, the lipase of Bufo melanostictus were both very unstable at 60°C. It was also previously reported that the amylase and lipase of Rana esculenta were unstable at 50°C (Scapin and Lambert-Gardini, 1979). In both species, lipase was less stable than amylase. The amylase was similar to the alkaline protease of the same toad in having a Table 3. The specific activities ~ moi oleic acid hr - l m g - ' protein- z) of lipase in the tissues and lumen of various parts of the digestive tract of Bufo melanostictus Region of gut Oesophagus Stomach Anterior small intestine Middle small intestine Posterior small intestine Rectum Pancreas
Tissue
Contents
448.5 + 17.5 423.0 _+43.8 188.5 + 7.6 369.9 + 13.3 265.1 + 15.1 28.8 +_ 1.8 423.6 + 17.6
482.1 _+ 10.0 457.4 + 15.8 152.5 + 11.3 409.5 + 10.1 430.0 + 20.0 394.5 + 14.9
portion of it very stable at high temperature (Teo et al., 1990). It is thought that there are perhaps two components of both the alkaline protease and amylase, one of them being highly thermostable. A similar observation was made in the sucrase of Drosophila melanogaster (Marzluf, 1969) and the amylase of the green mussel (Sahapathy and Teo, 1992). In the latter, the 50% inactivation time at 50°C was only 4.4 min and so it is more thermolabile than the amylase of Bufo melanostictus. It was quite surprising that the gastric mucosal extract had such a high total lipase activity although much of the lipase in the mucosa was proabably not secreted into the gastric lumen. Although the oesophageal mucosa did not have such a high total lipase activity, it seemed that much of it was secreted into its lumen, as judged by the relatively high activity level in its contents. The latter was one of the highest activity levels recorded. However, the contents of the oesophagus probably do not stay long in it but pass quickly, together with food, into the stomach where the acidic conditions would not allow lipase to be active. The lipase present in the gastric lumen might become denatured in the acidic conditions before it reached the anterior small intestine. That might explain why the latter had the lowest level of lipase activity in its lumen. Based on this argument, the digestion of fat seems to depend a lot on lipase supplied by the pancreas and the small intestine. The rectal mucosa had a low level of lipase activity but its contents had lipase activity comparable to that of the middle and posterior small intestine and so some of the lipase present in its lumen probably had its origin in the more anterior part of the intestine, as well as the pancreas. The contents of the anterior small intestine had the lowest total units of lipase, the lowest units of lipase per ml of contents and also the lowest specific activity of lipase. The anterior small intestine is least important in the digestion of lipid. The important regions in the digestion of lipid seemed
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Table 4. The specificactivities (mg maltose hr-m mg protein-m) of amylaseon starch and glycogen in different parts of the gut of Bufo melanostictus Stash Region of gut Oesophagus Stomach Intestine 1 Intestine 2 Intestine 3 Rectum Pancreas
~yco~n
Tissue
Contents
Tissue
Contents
11.80 __ 0.09 4.86 + 0.19 7.88 4- 0.24 7.894-0.26 10.39 4- 0.93 11.964-0.20 17.15 4- l . l l
1.85 4- 0.09 1.02 4- 0.05 1.86 + 0.10 1.49 4- 0.05 2.39 + 0.09 1.594-0.05
1.22 4- 0.13 0.35 4- 0.04 1.23 4- 0.28 1.14 __ 0.30 1.45 4- 0.33 1.784-0.49 40.16 __ 3.76
3.21 _+0.41 1.73 __ 0.53 5.94 4- 0.24 1.91 4-0.37 5.06 4- 0.60 2.71 4-0.41
Intestine 1: anterior small intestine. Intestine 2: middle small intestine. Intestine 3: posterior small intestine.
to be the middle and posterior regions of the small intestine. The contents of rectum also had high lipase activity but as it is the last region of the intestine, the food probably does not remain in it long enough for sufficient absorption of products of digestion to take place across its walls. Although the oesophagus had high activities of lipase and moderate activity of amylase in its contents, it probably has a small role in digestion as foods normally pass through it directly into the stomach. Although the pancreatic extract had one of the highest specific activities on lipid, and its total unit of lipase per ml of enzyme was also the highest, the total of units of lipase in the whole pancreas was lower than that of stomach and oesophagus and also not very much higher than that of the mucosal extracts of the anterior and middle small intestine. Its total of units was only half of that present in the entire small intestine. Thus it may be concluded that the contribution of the pancreas to lipid digestion was not the most important one. The anterior small intestine, on the other hand, had the highest activity on glycogen in terms of the total units per organ and total units per ml of enzyme extract and specific activity. All these indicate that the anterior small intestine is most important in the digestion of glycogen. The posterior small intestine is probably only second to the anterior small intestine in its importance to the digestion of glycogen. The higher activities on glycogen than on starch found in the small intestine and rectum might be explained by the input of amylase from the pancreas. The lumen
of rectum would also receive input from the small intestine. But the activities of extracts from the contents of the oesophagus and stomach were also higher on glycogen than on starch. This either indicates regurgitation of enzymes from the anterior small intestine to the stomach, and even oesophagus, or the amylase isozyme which showed higher preference for glycogen as a substrate was secreted in larger proportions (out of its proportion present in mucosa of these two regions) in response to the diets of the toads. Pancreas is the most important source of amylase, with glycogen as the substrate. Its total activities far exceed those of all other parts of the gut added together and its specific activity, as well as activities per ml of enzyme extract, far exceed those of any other parts of the digestive tract. The contribution of the pancreas to the digestion of starch is far below its contribution to the digestion of glycogen. With regards to the activities on starch and lipid, the pancreatic extracts maintained the same levels of activities even after 5 days of starvation. The amylase activity of the small intestine was only slightly affected by starvation after 14 days. There was only a slight difference in its activity at the two different temperatures. On the other hand, lipase activity was much affected by both starvation and temperature. It had a greater dependence on these two factors than proteases (Teo et aL, 1990). In Rana esculenta, pancreatic lipase was not affected by starvation but pancreatic amylase was much affected, as its activity fell by 39% (Scapin and Lambert-Gardini 1979). In
Table 5. Effect of starvation on the enzymic activities [mean (SE)] in Bufo raelanostictus Normal room Enzyme (source) Starved for 5 days Amylase (Pan) Amylase (Int) Lipase (Pan) Lipase (Int) Starved for 14 days Amylase (Int) Lipase (Int)
Fed
11.97 (0.91) 72.8 (8.3)
Amylase:/tM maltose hr -I. Lipase:/~M oleic acid m i n - k Pan: pancreas; lnt: small intestine. Normal room: 28 + 3°C. Air-conditioned room: 24 + 3°C.
Starved
9.24 (0.64) 49.1 (4.1)
Air-conditioned room Fed
Starved
11.96 (0.15) 9.74 (0.72) 147.8 (12.0) 68.25 (3.8)
11.93 (0.12) 8.50 (0.44) 141.0 (8.8) 34.50 (1.90)
9.52 (0.68) 51.8 (6.8)
8.82 (0.68) 12.8 (1.9)
Toad amylase and lipase the bull frog, pancreatic amylase fell by 40% and intestinal amylase decreased by 70% after animals had been starved for 1 week; in the leopard frog, the respective figures were 37 and 60% (McGeachin and Welbourne 1971). It is interesting that keeping toads at two different temperatures, with only a small difference of 4°C, resulted in differences in the activities of both amylase and lipase in the intestine. The difference was even more spectacular in the case of lipase which showed a decrease of 29% activity in the fed toads and 74% in the starved toads at the lower temperature. It may be interesting to point out that the amylase activity is not only less affected by starvation but is also less affected by environmental temperature, since the decrease in activity was less drastic when toads were kept at a lower temperature. It is not certain whether this has anything to do with the fact that toads may depend heavily on glycogen as a source of energy for general motor activities. It would be an advantage to the toads to have a good supply of amylase at any time so that when food is available, the enzyme is there in sufficient quantities to digest it in order that the source of energy for muscles be readily available. REFERENCES
Bernfeid P. (1955) Amylases, ct and ft. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 149-158. Academic Press, New York.
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Berry P. Y. and Bullock J. A. (1962) The food of the common Malayan toad, Bufo melanostictus Schneider. Copeia 1962, 736-741. Kampen P. N. van (1923) The Amphibia of the IndoAustralian Archipelago. E. J. Brill, London. Marzluf G. A. (1969) Studies of trehalase and sucrase of Drosophila melanogaster. Archs Bioehem. Biophys. 134, 8-18. McGeachin R. L. and Welbourne W. P. (1971) Amylase in tissues of the bullfrog, Rana catesbeiana and the leopard frog, Rana pipiens. Comp. Biochem. Physiol. 38A, 457-460. Myrtle J. F. and Zell W. J. (1975) Simplified photometric copper-soap method for rapid assay of serum lipase activity. Clin. Chem. 21, 1469-1473. Reeder W. G. (1964) The digestive system. In Physiology oJ Amphibia (Edited by Moore J. A.), pp. 99-149. Academic Press, London. Sabapathy U. and Teo L. H. (1992) A kinetic study of the or-amylase from the digestive gland of Perna viridis L. Comp. Biochem. Physiol. 101B, 73-77. Scapin S. and Lambert-Gardini S. (1979) Digestiveenzymes in the exocrine pancreas of the frog Rana esculenta. Comp. Biochem. Physiol. 62A, 691~597. Smith M. A. (1930) The Reptilia and Amphibia of the Malay Peninsula. Bull. Raffles Mus. Singapore 3, 1-149. Teo L. H., Chen T. W. and Tan L. L. (1990) The proteases of the common Malayan toad Bufo melanostictus Schneider. Comp. Biochem. Physiol. 96II, 715-720. Teo L. H. and Woodring J. P. (1985) Digestive enzymes in the house cricket Acheta domesticus with special reference to amylase. Comp. Biochem. Physiol. 82A, 871-877. Vonk H. J. and Western J. R. H. (1984) Comparative Biochemistry and Physiology of Enzymatic Digestion. Academic Press, London.