The Arginase Activity of the Tissues of the Earthworms Lumbricus Terrestris L., and Eisenia Foetida (Savigny)

Cohen & Lewis (1950) found that the arginase activity of the tissues of Lumbricus was related to the output of urea by intact worms, being much higher in fasting than in feeding individuals. This implies that the earthworm may produce excretory urea by the Krebs-Henseleit mechanism (Krebs, 1934) or by some variant of it. In general invertebrates are not ureotelic and it has been suggested (Hunter & Dauphinee, 1925; Baldwin, 1953, pp. 305, 321) that they lack enzymes for the Krebs-Henseleit mechanism. Earthworms therefore are particularly interesting in this connexion.

Living Eisenia foetida produce less urea per unit weight than Lumbricus, when fasting, and show a smaller change in its output between the fasting and feeding conditions (Needham, 1957), so that the species provides suitably contrasting material on which to extend the experiments of Cohen & Lewis. The arginase activity of homogenates of its tissues, i.e. the rate of liberation of urea from added arginine, has therefore been measured, under fasting and feeding regimes. The tissues of Lumbricus have been studied under the same conditions, in order to compare the species differences in arginase activity with those in urea output by the intact worms (Needham, 1957).

Cohen & Lewis found a very high arginase activity in the gut tissue of Lumbricus and very low activity in the body wall, or ‘carcass’, and the two components therefore have been separated for study in the present experiments also.

For each experiment a single individual of Eisenia was used, or a portion of Lumbricus of comparable weight, between 200 and 300 mg. The worms were killed by immersion in 10% ethanol for 5 min. at room temperature. They were freed of superficial fluid and mucus, and weighed. The body wall was opened longitudinally and pinned out. The septa were broken down and coelomic fluid absorbed with filter-paper. The gut was opened along its length and the contents mopped away with damp filter-paper. It was then dissected out from the body wall and both placed on dry filter-paper for 1 min. to remove adherent fluid, before weighing. In fasting Lumbricus gut and body wall weighed 12·6 and 48·7%,respectively, of the total wet weight, and 11·1and 52·9%, respectively, in feeding individuals. For Eisenia the four values, in the same order, were 13·4, 53·2, 15·0 and 49% 0%. The regime differences are in opposite directions in the two species.

Each tissue was homogenized by hand in a tube of the Potter type. The gut was completely homogenized within 1 min. but the body wall, particularly of Lumbricus, required 3 min. before all solid fragments were broken up. Small quantities of earthworm Ringer (Pantin, 1948) were added to the tube, and the suspended homogenate was forced out into a tared tube by the plunger. Grinding and suspension were continued until all of the material had been ejected in this way. 1 ml. of 0·02M cobalt chloride was added, as co-factor for the arginase, followed by 4 ml. of M/4 disodium hydrogen phosphate as buffer. As antibiotics, streptomycin and penicillin were added in amounts designed to give 1 mg. (735 units) and 0·3 mg. (500 units), respectively, per ml. of the final preparations. The volume was made up to 8 ml., which was thoroughly mixed and divided equally between two incubation tubes, one to serve as control. The tared tube was washed out with 8 ml. of Ringer, shared equally between the two incubation tubes. Finally, 2 ml. of a 2% solution of the substrate, L-arginine monohydrochloride, were added to the experimental tube and well mixed ; 2 ml. samples were then withdrawn from both tubes for a zero-hour estimation.

The tubes were lightly stoppered and incubated for 6 hr. at 23° C., the temperature previously used for the study of urea output by intact worms (Needham, 1957). The average pH of control and experimental tubes was 7.8 and 7.6, respectively. The small difference between the two is of little consequence since there was relatively little activity in the control tube. Arginase activity is maximal at a higher pH than this, but destruction of the enzyme also is more rapid.

The urea liberated was estimated as ammonia, after incubating 2 ml. samples for 20 min. at 30° C. with 10 mg. of soya-bean meal as a source of urease. The mixture was well shaken to ensure adequate solution of this enzyme. The resulting ammonia was estimated by the Sorensen formaldehyde method ; any ammonium and amino groups present also would be included in the estimate, which therefore may be called the AAU-nitrogen content of the sample. The difference in content between experimental and control tubes may be taken as due to urea-nitrogen released from the added substrate, together with known amounts of amino-nitrogen introduced by the substrate. These are the α-NH₂ nitrogen of the arginine and the ω-NH₂of the ornithine released, the three guanidino nitrogens of arginine being Sorensen-negative. It is clear that the ornithine ω-NH₂ nitrogen should be exactly one-third of the total nitrogen released from arginine, but that as a check it could be estimated directly, by omitting the urease treatment. This was done on one sample. The zero-hour estimate for the experimental tube, after deducting the control reading, gave a check on the concentration of arginine α-NH₂ nitrogen and was used as the measure of this.

From the results ‘specific’ values were computed, i.e. the initial content of AAU-nitrogen per unit weight of total nitrogen in the homogenate of the control tube, and the weight of urea nitrogen subsequently released per hour per unit weight of total nitrogen in the experimental tube. Cohen & Lewis (1950) expressed some of their results on the basis of wet weight, and present estimations, on eight samples of each tissue, showed that total nitrogen as a percentage of wet weight varies little, except in fasting Eisenia. It was estimated by the Kjeldahl method, using a Markham semi-micro apparatus for the steam-distillation. In the gut and body wall of fasting Lumbricus the values were 2·20 and 2·55% of wet weight, respectively, and 2·16 and 2·32 %, respectively, in feeding individuals. For Eisenia the four values in the same order were 3·21, 2·90, 2·43, 2·21%. The values are seen to be consistently higher in the tissues of fasting worms than in the corresponding tissues of feeding ones, and in those of Eisenia than in the corresponding tissues of Lumbricus. The gut/body wall ratio was greater than unity in Eisenia but less in Lumbricus.

In this series of experiments 216 preparations were studied. In addition 164 were used for an earlier series in which thymol was used as antiseptic. The results for that series were in accord with those of the present series but will not be considered in detail.

The results are summarized in Table 1. Those for Lumbricus are in close agreement with those of Cohen & Lewis (1950) both in general features and in the actual values for specific arginase activity given in their Table 2. Activity was higher in fasting than in feeding worms; in gut tissue the difference was significant with a value of P considerably less than 0.01. In the body wall activity was low and the régime difference was not statistically significant during this period. Specific activity in the gut was around thirty times that in the body wall. The difference in activity between corresponding tissues of fasting and feeding worms was less than that recorded by Cohen & Lewis but their feeding worms were all direct from the field, whereas those of the present experiments were laboratory-fed for a period of weeks.

In Eisenia, also, specific activity was higher in gut than in body wall, and under both régimes the difference was significant beyond the 1 % level. The difference was considerably less than in Lumbricus, however. In virtue of its relative bulk the body wall therefore makes a major contribution to the total arginase activity of Eisenia.

For both tissues the fasting/feeding difference in specific activity was less than in Lumbricus and was not statistically significant. Although more consistently higher in fasting worms when calculated on a wet-weight basis, the difference still was not significant. The régime difference in specific urea output by living worms similarly was found to be less than in Lumbricus (Needham, 1957).

The specific activity of the gut of fasting Lumbricus was significantly greater than that of Eisenia, and the species difference in urea output by living fasting worms must depend mainly on this tissue, since the specific activity of the body wall was significantly higher in Eisenia, under both régimes. In feeding, as in fasting,.worms the activity of the gut of Lumbricus was greater than that of Eisenia, but here the significance of the difference was not beyond the 2 % level. Allowing for the relative bulk of the body wall, the total activity of the tissues therefore is greater in feeding Eisenia than in feeding Lumbricus (p. 780), and this is true of urea output by living worms.

For the gut and body wall, respectively, of fasting Lumbricus the amounts of ornithine ω-NH₂ nitrogen released were 43·6 and 39·2% of the total Sorensen-nitrogen released from added arginine, and for feeding worms 42·4 and 36·7%, respectively. The four values for Eisenia, in the same order, were 38·1, 40·8, 37·8 and 42·1%. These are all greater than the 33·3 % expected (p. 776) and for the tissues collectively the excess no doubt is significant. That for the gut of fasting Lumbricus gave a value of P less than 0.01 and that for the body wall of feeding Eisenia a value between 0·01 and 0·02.

A possible explanation is that the tissues have a slight urease activity. This enzyme is not uncommon in invertebrate tissues (Prosser et al. 1950, p. 203; Baldwin, 1953, p. 270). The activity varied between 5 and 17% of the arginase activity of the corresponding tissues, and this relative activity seems reasonable in animals which excrete a substantial proportion of urea as such. The relative activity was sufficiently uniform to give specific activities with much the same tissue, regime and species differences as for arginase activity, which would be consistent with a normal metabolic association between the two.

It is seen that the percentage excess was greater in the body wall than in the gut of Eisenia, but conversely in Lumbricus. A similar inverse relationship between the two species was seen (p. 777) for the nitrogen contents of the two tissues, the value being relatively low where the present activity was high. No functional association between this activity and low nitrogen content is necessarily implied, though the other species differences encountered in this work did show a different pattern. The species difference in the pattern indicates that the activity is genuine and not due to a technical factor.

This includes not only amino + ammonia + urea nitrogen present in, or produced by, the tissues themselves (p. 776), but also some due to the added urease and antibiotics, mainly to streptomycin. These extraneous components were estimated on samples from ‘bank’ tubes, i.e. control tubes lacking the tissue homogenate, and were deducted from the control tube values for the purpose of this section. The latter were small, and so subject to a considerable percentage error of measurement, but the residual values merit brief consideration. They show features which might justify more specific investigation.

The initial specific content of AAU-nitrogen of the control tubes (Table 1) showed a general proportionality to the arginase activity of the tissue, and was equivalent to about an hour’ s activity of the enzyme in the experimental tubes. This nitrogen therefore may originate, in part at least, from prior activity of arginase, in vivo, or from some correlated activity. Cohen & Lewis (1950) found that the urea content of the tissues of Lumbricus in fact increased, as the enzyme activity does, during fasting. Like arginase activity, AAU-content was significantly higher in the gut than in the body wall of both species, under both regimes. However, the differences were smaller than for arginase activity. Again, in general specific AAU-content was higher in the tissues of fasting worms than in the corresponding tissue of feeding worms, though none of these régime differences was statistically significant. Clearly the correlation is only partial and there were other differences from the pattern of arginase activity: for instance, not only in the body wall but also in the gut it was greater in Eisenia than in the corresponding tissue of Lumbricus. None of these species differences was statistically significant but this may be due to the large error of measurement and to the difference in one being partly offset by that in another component. The total AAU from the body wall would be greater than that from the gut tissue in both species, under both regimes, whereas for arginase activity this was true only in fasting Eisenia. The presumption must be that the initial AAU-content reflects intermediate nitrogen metabolism rather more generally.

The present results for Lumbricus confirm those of Cohen & Lewis (1950), while those for Eisenia further strengthen their conclusion that the arginase activity of the tissues of earthworms is related to the excretion of urea by living worms. It has been seen that the relationship is manifested in a number of further details. Ideally arginase activity should be measured in living worms, or spontaneous urea excretion in tissue homogenates, in order to make a critical comparison between them. It is possible, from the present results, to compute a total arginase activity, per gramme of intact worm, but this ignores any possible activity of gut contents or of body fluid and also any systematic control of enzyme activity, and it would not be surprising if the results did not agree closely with figures for urea output by the corresponding living worm. The values obtained were 156, 85, 170 and 105 mg. of urea nitrogen per hour per 100 mg. wet weight of whole worm, respectively, for fasting and feeding Lumbricus, and fasting and feeding Eisenia. Apart from that for fasting Lumbricus, the figures in fact bear similar proportions to the corresponding urea outputs by living worms, 129, 41, 73 and 48 mg. of urea nitrogen per gramme per day (Needham, 1957). The particularly high output of urea by living fasting Lumbricus therefore may depend as much on systemic factors as on the high intrinsic activity of arginase in its gut tissue. There is in fact evidence for a systemic control of urea output (Needham, 1958).

Cohen & Lewis (1949) found also that the feeding of arginine to Lumbricus, and to a lesser extent that of citrulline, caused an increase in urea excretion, so that the enzyme presumably does function in normal urea excretion, in vivo. When the common amino acids were fed along with citrulline urea output was increased more than by citrulline alone and this probably implies that part of the urea, at least, comes from the deamination of amino acids in general. Abdel Fattah (1954) obtained an increased urea excretion by feeding the common amino acids alone. Cohen & Lewis failed to demonstrate this, perhaps because their animals had insufficient indigenous citrulline, but there is also the complication that, when feeding, Lumbricus excretes mainly ammonia and not urea, so that the former is probably the final product of deamination of any amino acids given as food. Possibly for the same reason they found that ornithine fed together with other amino acids likewise caused no increase in urea excretion. These authors further found that ornithine alone, in contrast to citrulline alone, caused no increase. This may indicate that, in the control of urea excretion, arginine synthesis may be halted at the ornithine, but not at the later, citrulline stage.

The arginase activity found in the body wall of Lumbricus is low enough to prompt the suggestion of Cohen & Lewis that it is due merely to contamination from the gut during dissection. However, this cannot be true in Eisenia where the total activity measured in the body wall is virtually equal to that of the gut ; the activity associated with the body wall therefore may be intrinsic in Lumbricus also. Much of the nephridial tissue remains attached to the body wall and may be responsible for the activity measured. If so, then there is an anatomical analogy with the vertebrates, of which the kidney tissue similarly possesses some arginase activity, and the specific activity is less than that of the liver (Hunter & Dauphinee, 1925), which is a gut appendage. The chloragogen tissue of earthworms has some resemblance to the digestive gland tissue of Crustacea and Mollusca, and Abdel Fattah found a particularly high urea content and arginase activity in the chloragogen tissue.

There are general grounds, as well as some evidence from the present results, for believing that much of the AAU-nitrogen originally present in the homogenates is related to the excretion of the three components by the living animal. This also can be tested further by computing the corresponding values for whole worms and comparing these with the AAU-excretion previously recorded for living worms (Needham, 1958). The values obtained, 153, 146, 385 and 213 μ,g. per 100 mg. wet weight of whole worm, respectively, of fasting and feeding Lumbricus and fasting and feeding Eisenia, are very near to the amounts of AAU-nitrogen excreted per gramme per day by living worms, viz. 159, 95, 346 and 205 p.g. The largest percentage discrepancy is that for feeding Lumbricus, which therefore appears to excrete less of the AA-components than indicated by the activity of its tissues (the urea component was shown above to agree with expectation, in contrast to that of fasting Lumbricus). Like those for AAU-excretion by the living worm, the values are considerably higher in Eisenia than in Lumbricus and for both properties the fasting/feeding ratio shows a comparable difference between the two species.

The conclusion that the initial AAU-content is related to normal nitrogen excretion could be tested by specific experiments designed to measure the ammonia-producing activity of the tissues, for instance, the glutaminase I activity, using glutamine as substrate, and the proteolytic activity, using a suitable protein as substrate.

The properties studied are seen to vary less between tissues and régimes in Eisenia than in Lumbricus. The latter therefore is probably the more highly evolved genus.

It is interesting that the vertebrates, which as a group are ureotelic (Brown & Cohen, 1960), use creatine phosphate and not arginine phosphate as the phosphagen of their muscles and that earthworms also possess a phosphagen, lombricine (Rosenberg & Ennor, 1959), which is not based on arginine. There is a possibility, therefore, that muscle arginine might be excessively destroyed in ureotelic animals, which in consequence have exploited a different phosphagen. Possible objections to this view are that little arginase is usually present in muscle tissue and that molluscs, such as Helix, appear to excrete considerable urea but yet to use arginine phosphate as their phosphagen.

I am indebted to Prof. R. B. Fisher for very helpful discussions on aspects of the work, to Drs M. G. Ord and L. A. Stocken for references, and for their comments on the manuscript, and to Mr P. L. Small for a supply of earthworms.