ABSTRACT
The enzyme arginase which occurs in various organs of different animals was first discovered by Kossel and Dakin (1904) in the mammalian liver. Clementi (1916, 1918, 1922) found arginase in the liver of mammals, amphibians and fishes and the turtle, but he did not find any in the liver of birds and of the majority of reptiles. He found the enzyme to be present in the kidney of birds. Hunter and Dauphinee (1924) found a considerable amount of arginase in the liver of mammals and fishes. They also established the presence of arginase in the kidney of mammals and fishes, but usually in an amount smaller than that present in the liver. In the organs of fishes other than the liver, heart and kidney, the distribution of arginase appeared to be variable. In mammals these workers did not find arginase in any organs except the liver and the kidney, while in birds they could find it only in the kidney.
Edlbacher and Röthler (1925) made an extensive study of arginase content in the domestic fowl and in certain mammals. They found that arginase was present in the liver, kidney and testis of both mammals and birds. Further they detected the presence of the enzyme in the placenta and thymus of some mammals. These authors were the first to find any arginase in the testis, and they also found arginase in the liver of birds, which all the previous workers failed to detect. They established that in mammals the organ richest in arginase is the liver, while in birds it is the kidney. The relative quantity of the enzyme present per gram of body weight is far greater in mammals than in birds. They also demonstrated that the arginase value (which is the number of units of arginase per gram of body weight) was higher in the males than in the females.
The present piece of research was undertaken at the suggestion of Dr Crew, with the view to testing and extending the work of Edlbacher and Röthler. It is intended to present more data on the conflicting question of the presence and absence of arginase in the liver of birds and also to make a thorough study of its presence in different organs of the fowls, and its relation to sex.
The material used for the work consisted mainly of the Bantam breed of fowls. A few data refer to other breeds of fowls and to mammals.
The method used for detection and estimation of arginase throughout this experiment is that used by Edlbacher and Röthler, since it is the most up-to-date and claimed to be most accurate. Arginase can be extracted with water and dilute acetic acid, and can be separated from this solution by ammonium sulphate and alcohol and ether. It can also be extracted with glycerine. It is claimed that the extraction with water is not so effective as that with glycerine. But so far as the organs of birds are concerned, where the quantity of arginase present is usually small, there was found to be little difference between the two methods of extraction. In quite a number of cases where control experiments were carried out with glycerine extract to test the presence of the enzyme where water extract failed to detect any, the results were again negative. Throughout this piece of work the extraction has been carried out with water. The one advantage this has over the method of glycerine extraction is that the organ can be used fresh, and can be handled with more accuracy. One gram of the organ to be tested was taken and ground with sufficient quartz-sand until a homogeneous paste was obtained. 25 c.c. of distilled water were then added, and the mixture well mixed; after standing for five minutes the extract was separated from the coarser sediment. A known quantity of this water extract was then taken for the determination of quantity of arginase present.
Edlbacher and Röthler found that the maximum activity was obtained when the reaction of the medium was 9·5 pH at a temperature of 38° C. For each estimation a definite quantity of 10 c.c. of the water extract was taken to which were added 5 c.c. of glycocoll-NaCl-NaOH of pH 9·5, then 10 c.c. of 1 per cent, arginine carbonate solution, and a few drops of toluol. This was corked and placed in a thermostat at 38° C. for one hour. At the end of one hour the ferment was destroyed by boiling for about 15 minutes. In each case control experiment without arginine carbonate was carried out. Next the solution was cooled, and the pH brought to 7 by the addition of phosphate buffer solution. The mixture was hydrolysed with ureas (soya bean powder) and the evolved ammonium driven to a known volume of N/50 H2SO4 by passing a current of air through the apparatus. Finally the ammonium formed from the urea separated from arginine carbonate was estimated by titrating the excess of H2SO4 with N/50 NaOH. For a comparative study it is desirable that some definite unit be chosen for measuring the amount of the enzyme present—for this I have chosen the unit adopted by Edlbacher and Röthler. The unit of arginase is the quantity required to produce from 10 c.c. of a 1 per cent, arginine carbonate solution to which has been added 5 c.c. of glyçocoll-NaCl-NaOH of pH 9-5 at 38° C. during the period of 60 minutes such an amount of urea which on being treated with urease produces 0·34 mg. of ammonium.
RESULTS
In birds, kidney is the organ richest in arginase. This finding is in conformity with other workers. Next to the kidney, testis stands high for the presence of the enzyme. The exact amount varies in different individuals, but at the same time there is always a significant amount present in the materials examined. This finding is in accordance with the work of Edlbacher and Röthler but in contrast to that of Clementi, Hunter and Dauphinee. Clementi, Hunter and Dauphinee did not find any arginase in the liver of birds, while Edlbacher and Röthler found quite an appreciable amount. In my material the quantity present in the liver, however, is smaller than in either the kidney or the testis. Out of the livers of 32 birds examined, only 11 showed any trace of the enzyme, and the amount present was very small. It may be that the enzyme is not universally present in the liver of birds, or that the very small quantity which is usually present has escaped extraction in the above 21 out of 32 cases.
REPRODUCTIVE ORGANS
As was mentioned above, there is a certain amount of arginase present in the testis and also in the vas deferens. No arginase was ever found in the ovary, oviduct, or in the ova. This indicates that the presence of arginase in the reproductive organs is a sex-dimorphic character.
OTHER ORGANS
Several determinations with thyroid, thymus, heart, spleen, pancreas and blood of fowls did not reveal the presence of any traces of arginase. The organs of 17 birds were examined fully for quantitative estimation of the enzyme, and the results obtained have been tabulated in Table I.
The results obtained have been divided into two sections (a) and (b) according to the sex of the birds. In both the sections, the figures have been arranged according to the ascending order of the body weight of the individuals. It is evident from the figures that the average arginase value of the males is higher than that of the females. The value for females (0·107) is 69·9 per cent, of that of males (0·153). The average number of units per gram of the kidney of the males, which is 15·65, is also higher than that of the females, which is 11·71. This latter is 74 ·8 per cent, of the former. From the above figures it is proved that there is a sex-dimorphism with regard to arginase content in fowls, and that the higher arginase value is associated with maleness.
ORIGIN OF THE ENZYME
It has been shown that there is no arginase in the ovary or in the mature ova, but there is a significant amount in the testis and vas deferens. The question then arises how the new individual comes to possess the enzyme. It is probable that the fertilised ovum gets its arginase from the male through the spermatozoa. A few eggs were examined which showed no trace of the presence of the enzyme. This is perhaps due to the small amount of the enzyme introduced by the spermatozoa. A few newly hatched chicks were examined and showed the presence of a significant amount of the enzyme. Three chicks were studied for quantitative estimation; the results obtained are given below. The testes, owing to their small size, were not examined.
It is seen that the enzyme arginase which was not detectable in the unincubated eggs can be estimated as soon as the eggs are hatched. Clementi found arginase in human embryo as early as four months. It would be of interest to determine after how many days of incubation the eggs show an appreciable amount of the enzyme and also whether it has any relation to the time of sex-differentiation.
Apart from the fowl material, only a few specimens of liver and testis of mammals were examined. With regard to the testis of a cat and a rabbit, it can be stated that both exhibited the presence of arginase which is contrary to the finding of Hunter and Dauphinee, but in agreement with those of Edlbacher and Röthler.
As regards the function of the enzyme, very little is as yet known except that it hydrolyses arginine into ornithine and urea. As the result of his researches, Clementi, who did not find any arginase in liver of birds, but found it in the liver of mammals, amphibians and fishes, enunciated the doctrine that arginase is present in the liver of all those animals which have what he calls a “ureotelic” metabolism, that is in which urea is the final product of protein degradation, and absent from the liver of those in which protein metabolism is “uricotelic”—ending in uric acid. Since the work of Edlbacher as well as the results of the present investigation do not show that arginase is totally absent from the liver of birds, it is rather difficult to accept his doctrine.
ACKNOWLEDGEMENTS
I have great pleasure in thanking Dr Crew for the help and encouragement he has given during the course of this study.