ARGINASE

ARGINASE

BY ERNEST BALDWIN, PH,D.~ (From the Biochemical Laboratory, Cambridge)

(Receiued July I, 1935)

CONTENTS

I. Introduction . . . . . 11. The occurrence of arginaac . .

(I) in the vertebrates - . . (2) in the invertebrates . . , (3) in the fungi and bacteria . ,

111. The functional importance of arginase (I) in ureotelic organisms . . (2) in uricotelic organisms . . (3) in growth . . . .

IV. Summary . . . . . . References . . . . .

PAGE . . . 247 . . . 250

. . . 250

. . . 255

. . . 257 . 259 . 259 . . . 261

. . . 263

. . . 265

. . ' . 166

. .

. .

I. INTRODUCTION

WHILE the ideally comparative review would embrace all the known forms of life, the limiting factor of space must necessarily preclude the attainment of this ideal, and the scope of this article has therefore been restricted to animals, fungi and bacteria. It is proposed to discuss the biological significance of arginase along rather broad lines in preference to giving an encyclopaedic account of the properties and possible functions of the enzyme. From a considerable mass of literature it has therefore been necessary to select for special consideration the papers which bear upon general rather than particular problems, and in this way it has been possible to trace some interesting correlations. Although a good deal of work has been devoted to the study of arginase as an intracellular enzyme, similar in many respects to other enzymes such as kathepsin and phosphatase, and although in view of our ignorance of intracellular processes in general this is a problem of great biochemical importance, it must be almost wholly neglected here. Again, the study of arginase in relation to the metabolism of tumours promises to lead to results of considerable importance, but this work too cannot be given more than incidental mention here. As key references to the literature of these aspects of the subject the papers of Waldachmidt-Leitz, Scharikova & Schlffner (1933),

The author wishes to express his gratitude to the Royal Commission for the Exhibition of 1851 for a Senior Studentship during the tenure of which this article was written.

248 ERNEST BALDWIN Klein & Ziese (1932 a, b, c, 1933) and the numerous publications of Edlbacher and his collaborators may be quoted.

Arginase is an enzyme which is very widely distributed among animal tissues. Many bacteria contain enzymes capable of decomposing arginine but not, as does mammalian arginase, with the production of ornithine and urea. I propose in what follows to reserve the name arginase for enzymes which catalyse the hydrolysis of d-arginine to give ornithine and urea. Arginase, in this proper sense, is one of the most specific enzymes known. The following substances, all of which are more or less closely related to arginine, have been shown not to be attacked by mammalian arginase :

Guanidinoacetic acid (Edlbacher, 1917; Edlbacher & Bonem, 1925 ; Dakin, 1907; Clementi, 1916).

B-guanidinopropionic acid (Edlbacher, 1917 ; Edlbacher & Bonem, 1925). y-guanidinobutyric acid (Thomas, Kapfhammer & Flaschentrager, 1923).l E-guanidinocaproic acid (Thomas, 1913). Guanidine (Clementi, 1916). Guanidinoglycylglycine (Clementi, 191 6). Creatine (Dakin, 1907; Clementi, 1g15b; 1916). Creatinine (Dakin, 1907). Agmatine (Edlbacher & Bonem, 1925). Tetramethylenediguanidine (Baldwin, 1934). a-6-di-(dZ-leucyl)-dZ-ornithine (Abderhalden & Sickel, 1929). Even Z-arginine is not attacked (Riesser, 1906; Edlbacher & Bonem, 1925).

Both d- and I-arginine are split if perfused through a surviving liver (Felix & Morinaka, 1923), but this would probably involve deamination and the production of the optically inactive argininic (a-oxy-6-guanidinovaleric) acid which, as Calvery & Block (1934) have shown, is almost as readily hydrolysed by arginase as is d-arginine.

This latter observation shaws that the presence of the free u-amino group is not essential for the activation of the molecule, though it might have seemed otherwise from the statement of Steib (1926) that dl-a-N-methylarginine is resistant to the action of arginase. On the other hand, the carboxyl group of the molecule must be free, for neither the methyl ester of arginine (Edlbacher & Bonem, 1925 ; Calvery & Block, 1934) nor the ethyl ester of argininic acid (Calvery & Block, 1934) is hydrolysed, while only 50 per cent. of the available urea is split from arginylarginine (Edlbacher & Bonem, 1925; Edlbacher & Burchard, 1931), and this comes from the arginine residue of which the carboxyl group is free. Probably the guanidine residue also must be unmodified, since arginine phosphoric acid is not attacked (Meyerhof & Lohmann, 1928) neither are 8-N-methylarginine (Thomas, Kapf- hammer & Flaschentrager, 1923) and 6-guanidino-N-caproic acid (Steib, 1926). The probability that both the carboxyl and the guanidine groups must be free is further emphasised by the fact that neither clupein nor clupeon is attacked by

These authors retract the earlier statement of Thomas (1913) that y-guanidinobutyric acid is hydrolysed by liver juice.

A rginase 249 arginase (Kossel & Dakin, 1904b), while clupeon can pass through the liver unchanged (Felix & Morinaka, 1923). These results are coilfirmed by the observations of Lieben & Lieber (1934).

Mammalian arginase has frequently been used as a reagent for the detection and estimation of arginine as, for example, by Meyerhof (1928), Hunter & Dauphinee (1930 a, b), Baldwin & Needham (1933), Arnold & Luck (1933) and others. It has usually been assumed in such cases that the specificity of arginase is absolute, but the application of a method based upon such an assumption to a material about which we know practically nothing is liable to give misleading results ; we do not know for certain that arginase is absolutely specific, nor do we know whether or not such material contains any potential substrate other than arginine. It is to be urged that when new substances related to arginine are isolated from invertebrate tissues, the action of arginase upon them should be immediately tested if there is any likelihood whatever that they may be attacked.

Work on the kinetics of the arginase-arginine system is difficult, since arginase is very labile under most conditions. It is fairly stable near the neutral point at ordinary temperatures, but with departure from neutrality is increasingly readily inactivated. Thus Hunter & Dauphinee (1933) found that at p H 5 or 10 about 50 per cent. of the activity of liver arginase is lost in only 10 min. at 37" C., and aboirt 75 per cent. in an hour. At pH values less than 4 or greater than 12 inactiva- lion is almost complete in 10 min. The optimum conditions of pH, temperature and time are interrelated variables, and in the case of arginase there is in practice an optimum neither of pH nor of temperature, but "at pH 9.8 the optimum for most conditions will be lower than 40" C. and for many lower than 30" C." according to Hunter (1934). Hunter & Morrell (1922) at first gave an optimum of p H 7.4, but later (1924) found the higher figure of 9.8. Edlbacher & Bonem (1925) found values of 9.5 and 9.8 at 26 and 38" C. respectively, while Hino (1926) obtained a figure of about 7.4. The more recent investigations of Hunter & Morrell (1933) make it certain that the optimum lies well on the alkaline side, near pH 9.8; the activity falls off sharply on the alkaline side on account of irreversible inactivation.

Gross (1920), working at p H 6.62 and 37" C., found that the velocity constants calculated from the equation for a monomolecular reaction fall off as the reaction proceeds, the reaction only going 70-85 per cent. towards completion. The composition of the reaction mixture was not altered in these experiments by the addition of fresh enzyme, and this appears to be because arginase is strongly inhibited by ornithine, though not at all by urea. According to Hino (1926) arginase is not affected by potassium bromide, iodide or cyanide or by quinine or atoxyl though, like most enzymes, it is destroyed by free iodine. Sodium fluoride inhibits and Waldschmidt-Leitz, Weil & Purr (1933) find that iodoacetate also inhibits, suggesting that the arginase system is in some way coupled to the oxidative systems of the cell. The results of Krebs & Henseleit (1932) indicate that the function of arginase in the intact cell is likewise dependent upon integrity of the cell structure and upon concomitant processes of oxidation.

Work on the activation of arginase was begun by Salaskin & Solowjew (1931,

250 ERNEST BALDWIN 1932 a, b), and soon taken up by many workers. But the results which have been obtained contain some peculiar contradictions. Thus Klein & Ziese (1933). have shown that whereas crude preparations are inhibited by molecular oxygen this is not true of purified arginase. Similarly, while crude preparations are activated by cysteine, glutathione and hydrogen sulphide, these compounds produce only a marked inhibition of purified preparations. Meanwhile Waldschmidt-Leitz, Weil & Purr (1933) had come to the conclusion that the -SH group is an essential cofennent and that “ ohne dies ist Arginase wirkungslos ”, a belief which was Coon given up however (Purr & Weil, 1934). With Klein & Ziese (1g33), Edlbacher, Kraus & Leuthardt (1933) point out that the behaviour of a given arginase preparation depends upon its previous history, and like Leuthardt & Koller (1934) point out that all the compounds which have an activating effect also possess reducing properties. They conclude that these activators, which include cysteine, glutathione, ascorbic acid, traces of iron, copper, manganese (Klein & Ziese, 1g35), and even the unphysiological substance hydrazine (Klein & Ziese, 1g33), act by a kind of Schutzwirkung, protecting, the enzyme against the inhibitory action of oxygen. The problem, which is full of complications-see, for example, Karrer & Zehender (1934)-cannot yet be regarded as fully understood, and more work is necessary to resolve the many factors which would appear to be involved. From the more purely biological viewpoint it seems likely that normal tissues always contain sufficient activatirig substances fo enable the enzyme to act at its full capacity within the cell, and that preparations in the form of a Brei probably give us a fairly reliable picture of the actual arginase content of any particular tissue.

11. THE OCCURRENCE OF ARGINASE

(I) In the wertehates The existence of a “urea-producing ferment” in the liver seems first to have

been suspected by Richet (1894, 1897). Kossel & Dakin ( I ~ o ~ u ) , however, were the first to show that urea is indeed present in such autolysates, and that it arises from arginine set free from the tissue proteins as a result of autolytic proteolysis. It was found that the enzyme, arginase, could be extracted with water and dilute acetic acid and precipitated from such watery extracts by means of ammonium sulphate, alcohol or ether, the precipitates showing a high degree of argininolytic activity. The enzyme, which was also present in press-juice, was found to catalyse the following reaction :

HN=C( NH, /NH* H,N=C\\

iH* NH 0 I

(CH3a - + ( HA3

LH(NH,) JH(NHJ LOOH dOOH

(arginine) (urea) (ornithine)

A rginase 251

The authors pointed out that autolysates of liver do not contain arginine, and that this must be because the organ in question contains arginase, and, referring to the absence of arginine from autolysates of thymus (Kutscher & Seemann, I ~ O I ) , intestinal mucosa (Kutscher & Seemann, 1902) and kidney (Dakin, 1903), wrote “das Fehlen des Arginins unter den Produkten der Autolyse lhst mit grosser Wahrscheinlichkeit auf die Gegenwart der Arginase in diesen Organen schliessen ”. Acting on this hint they proceeded (39046) to examine various tissues of the calf, ox and dog. Liver, kidney, lymph glands and intestinal mucosa of all three animals gave positive results, muscle a doubtful positive, blood, spleen and adrenal negatives. By far the richest source of arginase was the liver. Shortly after this Mochizuki & Kotake (1904), who could find no arginine in autolysates of ox testis, suggested that this organ also might contain arginase and Mihara (1911) showed that such is indeed the case.

Little further progress was made until Clementi, armed with a new method (1914a), extended the search for arginase to a series of vertebrates other than mammals (1914b). The titrimetric method of Clementi, which was based upon form01 titration, and which he afterwards modified (1922), was rapid and relatively sensitive; the major results which he obtained with it are summarised in Table I.

Class ___

Marnmalia

Aves

Reptilia (Sauria)

(0 hide) (CEeloma)

Amphibia

Pisces (Elasmobranchii)

(Teleostei)

Table I (Data from Clementi, 19146)

Species

Dog ox Pig Guinea-pig

*Rat +Monkey (Macaw rhcsus) +Mali GaUw domesticus Columba livia Turtur turtur F r i W clank LRcerta agilis Anguisfragilis Caronella dwtriaea Emys europae Ram esculenta R. temporaria Torpedo ocellata Raia clavata Perca fluviatdis Abramir brama Barbus fluviatilis

Additional data from Clementi (1922).

____- Liver

+ + + + + + + - - - - - - - + + t + + + + +

The livers of mammals, a reptile, the frog and several fishes contained arginase, which could not however be detected in the liver of birds. I t was found in the avian kidney and later (Clementi, 1915a) also in press-juices prepared from

252 ERNEST B A L D W I N mammalian kidney. Intestinal mucosa, spleen, testis, ovary and muscle from various animals contained no detectable amounts of the enzyme.

These findings led Clementi (1914b, 1 9 1 5 ~ ) to enunciate the rule that arginase is present in the livers of animals having a ureotelic metabolism (mammals, chelonian reptiles, fishes, Amphibia) but absent where the metabolism is uricotelic in character (birds, sauropsid and ophidian reptiles). Felix & Tomita (1923) found that arginine is rapidly broken down to urea and ornithine when perfused through a surviving mammalian liver, but not in the case of the bird. In passing it is interesting to notice that Clementi’s data ( 1 9 1 5 ~ ) reveal the fact that of the fish livers studied, those of elasmobranchs were much more active than those of teleosts, while arginase was also found in the only elasmobranch kidney examined.

The technique of formol titration has also been used by Edlbacher (1915) in another comparative study of vertebrate tissues, the results of which agree fairly well with those of Clementi. Tissue suspensions were usually employed, but in some of the experiments press-juices were used with confirmatory results. The most important data are summarised in Table 11. Mammalian thymus, spleen, testis

Table I1 (Data from Edlbacher, 19x5)

Class

Mammalia

Aves

Reptilia (Sauria)

Amphibia (Ophidia)

Species

Calf Dog Rat Cat Guinea-pig Fowl Pigeon Blindworm Viper Frog

____ Liver

brain and intestinal mucosa were inactive, and very similar results were obtained by Fuchs (1921) who found arginase in human liver but not in the thyroid, pancreas, adrenal, spleen or kidney.

The next substantial step forward was made by Hunter & Dauphinee (1924 a, b). Arginase was extracted from the tissues under examination by means of glycerol and the extract incubated with arginine under closely defined conditions of arginine concentration, pH, temperature and time. The relation between urea production and arginase concentration having been determined it was possible to express any given urea production in terms of an arbitrary unit of arginase concentration by reference to a standard curve. Urea was estimated by a colorimetric method elaborated by the same authors (1924~). The arginase activities, expressed as they were in terms of a purely arbitrary unit, had no absolute significance, but served nevertheless to give an excellent picture of the relative activities of different tissues. Table I11 presents the most interesting of the results; the numbers represent the

Arginase

Table I11

(Data from Hunter & Dauphinee, 19246; see text)

Cat ' Rabbit Hen Pigeon " Mud-turtle " Dogfish (S lus mMii7 Herring ( C g m pdlarii) Other telmsts

Mammalia

Aves

Reptilia (Chelonia) Pisces (Elasmobranchii)

(Teleostei)

0 0

rq'z 319 181

8-1 1 0

- 18.3 0 0

31 7

1-5

253

number of arginase units per C.C. of the various glycerol extracts, all of which were prepared in a strictly uniform manner. Numerous organs other than the liver and kidney were examined in the course of this work and, with a few exceptions, uni- formly negative results were obtained. But the heart and the muscles of the herring gave values of 8.8 and 0.9 units respectively, while the corresponding figures for the dogfish were 19 and 2.2 units. Furthermore, in the dogfish the only organs which were free from the enzyme were the brain, blood and ovum, although the latter contains a high concentration of arginase towards the end of development. The further researches of Hunter (1929) show that this wide dispersal of arginase in the tissues is characteristic of the elasmobranchs generally, since it was found both in the Batoidea and the Selachia. In contrast to these forms, the Holocephali, represented by the rat-fish, Chimaera colliei, appear to contain very little arginase, which is interesting since they are sometimes regarded as "a divergent and specialised offshoot from some primitive elasmobranch type " (Bridge, 1904). In Chimaera the liver and the heart contained at most only traces of arginase and the kidney only about 4 units per C.C. of extract.

Meanwhile the kinetics of the arginase-arginine system had received some attention, and Edlbacher & Bonem (1925) now worked at p H 9.5 in glycine buffer, decomposed the urea produced by means of urease, distilled off the ammonia set free and estimated it titrimetrically. Arginase was again found in mammalian (cat, mouse, dog, calf, guinea-pig, man) and in amphibian livers (frog), while bird livers (hen, pigeon) contained only traces, those of male birds containing more than those of females. Similarly the testis (cock, pigeon, ox, dog, guinea-pig) contained relatively more arginase than the ovaries (hen, pigeon, bitch) ; the testis of the calf resembled ovaries in arginase content. Mammalian kidneys contained traces of the enzyme (dog, rabbit, cat, mouse, guinea-pig) and the kidneys of birds also, but none could be demonstrated in the spleen (dog, cat, rabbit, mouse, guinea-pig, hen, pigeon), in the adrenal (guinea-pig) or intestinal mucosa (dog, cat, pigeon). Sendju (I 925), whose comprehensive paper seems to be undeservedly little known, reported entirely similar results, except that the kidney, spleen and pancreas, as well as the liver, of a turtle proved to contaiii arginase.

254 ERNEST BALDWIN Subsequent work has done little to modify these conclusions, though Ackermann

(1932) found that the kidney, muscle, and especially the spleen of the ox show a slight argininolytic activity. Blood also may bring about a slight hydrolysis of arginine v e i l & Russell, 1934), but in all these cases it is a little difficult to feel certain that the observed results are really due to arginase. On the other hand, Hino (1924) found pancreatic juice to be free from arginase, while Edlbacher & Rothler (1925b) again found no evidence for its presence in the spleen, thyroid, pancreas, intestinal mucosa or muscle of any of the species which they examined, though traces were found in the thymus (calf) and placenta (rabbit, guinea-pig). That arginase is present in the placenta is suggested also by Salaskin, Solowjew & Tjukow (1932), who find that urea arises from arginine during placental autolysis : according to Solowjew & Mardaschew (1932) the arginase-arginine system is entirely responsible for the urea which appears during the autolysis of liver. On the whole we are probably justified in concluding that, in general, significant quantities of arginase are only to be found in the liver, kidney and testis.

The way was now prepared for further quantitative studies, and Edlbacher & Rothler (1925~) devised a method whereby it was possible to obtain numerical values having some degree of absolute significance for the relative arginase activities of different tissues. They adopted the use of urease followed by distillation and titrimetric estimation of ammonia for determining urea production. A standard glycerol extract of calf liver was prepared, and increasing quantities of this were incubated with arginine under standard conditions, the amounts of urea formed being determined in the manner just indicated. It was found that the activity of the enzyme, as judged by the amounts of urea formed, was not proportional to the enzyme concentration but fell off markedly as the latter increased. This could probably be accounted for if the enzyme were inhibited by the products of its action (cf. Northrop, I~ZO), and it is known that arginase is strongly inhibited by ornithine (Gross, 1920). Nevertheless, provided that the conditions were kept constant, the form of the activity-concentration curve was the same whatever tissue was employed, so that the original curve for calf liver could be used as a standard of reference. It was only necessary to define some convenient unit of arginase concentration in order to be able to interpret the urea production in any given case in terms of the corresponding arginase concentration, provided always that the standard conditions were rigidly observed. The unit chosen was that amount of enzyme which liberated an amount of urea corresponding to 0.34 mg. ammonia in 60 min. when incubated with 10 C.C. I per cent. arginine carbonate and 5 C.C.

glycine buffer at pH 9-5 and 37 C., toluene being present as an antiseptic. By determining the amount of urea produced by a known amount of tissue under these conditions, the number of arginase units per gram of tissue (Edlbacher’s Arginase- w u t ) could readily be calculated. It was found that the kidney arginase of fowls gave an almost linear activity-concentration relation, though that of the duck and the dove resembled that for calf liver, and Edlbacher & Rothler attribute this to the absence from fowl liver of some inhibitor which they suppose to be present in all the other tissues. I t was necessary to construct a special standard reference curve

Arginase 255

for this special case and in addition to define another arginase unit. This was accordingly done, but for all practical purposes the “kidney unit’’ (A.N.E.) and the original unit (A.E.) are identical. This method was now used (Edlbacher & Rothler, 19qb) in a study of the relationship between sexuality and arginase content.

The fowl and several mammals were examined. In the males the liver, kidney and testis were analysed and in the female only the liver and kidney. It was shown that other tissues do not contain significant quantities of the enzyme, so that the total arginase content of the whole animal could be approximately evaluated. Table IV summarises the results, which are expressed in terms of arginase units

No. of exps.

2 4 4 5 4

Table I V (Data from Edlbacher & Rtithler, 19zsb)

Arginase unite per gm. body wt.

Males Females

66 5 0

38 29 77 72 43 60

61 2; 66 127 103

I Animal I DQB cat Rabbit Guinea-pig Rat

I Fowl I 2o I 0.363 1 0.277 1 63

Note that these data are not numerically comparable with those of Hunter & Dauphinee given in Table 111.

per gram of whole body. In each case it appears that the total arginase content of female is only about two-thirds of that of male animals, and this relation holds as well for the birds as for the mammals in spite of the very great absolute difference between the two groups. The same is true if individual organs are considered; this is shown in Table VII, the data of which are calculated from Edlbacher & Rothler’s data but are expressed ?n different terms, which will be explained later. Fujiwara’s results (1929), obtained with mice and guinea-pigs, are entirely in support of those just discussed, and the influence of sexual factors is also shown in that chicks contain less arginase per unit weight than do adult fowls, while the arginase content of the testis of the ox increases at puberty (Edlbacher & Bonem, 1925).

(2) In the inoertebrures Hunter & Dauphinee (1924b) came to the conclusion that “arginase is an

enzyme almost, if not entirely, peculiar to the vertebrates”, a belief which closely fitted the current views on the distribution of arginine itself. The relevant facts have been exhaustively reviewed by Hunter (1928), Baldwin (1933) and by Kutscher & Ackermann (1933); for the present it is sufficient to say that until recently arginine was thought to be almost universally distributed in the invertebrates but replaced by creatine in the vertebrates. This, together with the few data summarised in Table V, gave the situation a misleadingly watertight appearance.

256 ERNEST BALDWIN Table V. Arginase in invertebrates

Part analysed

Whole body Hepatopancreas

Hepatic caecae Hepatopancreas Digestive gland Hepatopancreas

,,

Species

Termite larvae Snail (He& pomutia) Crayfish (Astam fluviutilis) Starfish (Piscuter ochracea) Crab (Cancer productus) Clam (SaJCidonruc giganteus) Crayfish (Astam?)

Arginase Author

- Clementi, 1918 + ,, - Hunt:; & Dauphinee, 1924b - ,, . - Ackennann, 1932

--

-

- 9, ,.

As Clementi (193ob and earlier papers) pointed out, tne presence of large amounts of arginase among the vertebrates seems always to be associated with the production of urea, but Baldkin & Needham (1934) came to the conclusion that the snail, Helixpomatia, probably makes the urea which it excretes from exogenous arginine, thus resembling the hen (Clementi, 193zb). This raised the question as to how far the excretion of urea by invertebrates in general can be attributed to the action of a tissue arginase on ingested arginine, since most invertebrates excrete a certain amount of urea (Delaunay, 1927, 19311). It was therefore desirable to develop a very sensitive method for the detection and estimation of arginase and to carry out a survey of the invertebrate phyla, and this was begun by Baldwin (1935~) . Conditions were worked out under which the urea production per mg. of tissue was constant, provided that the total amount did hot exceed 4 mg. urea under the standard conditions ; the initial slope of the activity-concentration curve then gave the required arginase content in terms of urea per mg. tissue. Controls were carried out for preformed urea in the tissues and the solutions, urea being estimated manometrically by the method of Krebs & Henseleit (1932)~. This is at least IOO times as sensitive as the distillation method used by Edlbacher & Bonem (1925) and by Edlbacher & Rothler (1925 a, b). Baldwin's results were expressed in terms of the QH notation of Krebs & Henseleit (1932), i.e. as c.mm. urea-carbon dioxide per mg. dry tissue per hour, the temperature being 28 instead of 37" C. as used by Krebs. This fact is expressed by using the symbol QE. It seemed better to use this mode of expression than to add yet another to the list of arbitrary units already defined. The results of Edlbacher & Rothler (19256) can be expressed in the same terms, and some of them have been so recalculated and are given in Table VII. It is evident from Table VI, which presents the results obtained by Baldwin, that arginase is more widely distributed than was formerly supposed, and it would clearly be desirable to extend the observations to many other animals. The amounts of arginase present in organisms such as the crab and the starfish are very small, suggesting that it was probably on account of lack of sensitivity that some, at any rate, of the negative'results listed in Table V were obtained by the earlier workers. But the values for the terrestrial and fresh-water gastropods are very striking, for they are of the same order as those for mammalian livers. The possible significance of this will be discussed later.

Biological Reviews. a A similar technique has been used independently by Weil & Russell (1934) in their studies

of blood arginase. Their paper appeared shortly before the work of Baldwin (1935 a) was completed.

Arginase

I I Liver

257

Kidney I Testis

Table VI (Data from Baldwin, I935U)

9

2200 I450 1200 2200 19-

3

Phylum and class

6

7 16 95 16 42 36

Craniata: Mammalia

Echinodermata : Asteroidea

Arthropoda : Crustacea

Mollusca : Lamellibranchiata

Gastropoda

Species

Guinea-pig 0 (Cma)

Starfish (Asterius rubens)

Shore crab (Cmcim macnas)

Mussel (Mytilus eddis) Queen scallop (Pecten vperculmir) River mussel (Anodontu cygnaea) Whelk (Buccinuq undatum) Periwinkle (Littorinu littorea) Limpet (Patella wlguta) River snail (Vivipmw facciatus) Ram's horn Planorbis comeus) Pond snail (j!imnuea stugnulis) Common snail (Helix aopersa) Roman snail (Helix paratiu)

Black'blug (Ation &) (starved)

NO of specunena --_

2

3

8 8 8 2 16

8 12 24 7 2 3

1 0

QE

750

1'5

I5

0'0 0'0 2.8 0 0 0'0 0'0

719

635

6500

22.5

1235

1070 11'1

The organs analysed were the liver and the hepatic c a m e in the guinea-pig and the starfish respectively and the hepatopancreas in all other cases. The nomenclature of the non-marine forms follows Ellis (1926).

Table VZI. Arginase contents of some titsues, calculatedfrom the data of Edlbacher W R8thler (1925 b)

I d

Mammalia : Dog

Aves : Fowl

3300 2650 I 800 3900 32m

4

0

27 I7 77 16

6

8 20

I I

I 23 I 13

(The figures correspond to values and are calculated assuming a dry weight equal to 20 per cent. of the wet weight for all cases.)

( 3 ) In the fungi and bacteria There can be no doubt that many micro-organisms are capable of breaking

down arginine, but in general it seems unlikely that arginase, properly speaking, is concerned in the process. Shiga (1904) announced that arginase is present in yeast, but his technique was poor. Edlbacher (1g17), using the formol titration method, was unable to confirm Shiga's result, but it is of course possible that the two observers used different yeasts. Kiesel (1922) obtained agmatine and urea by

258 ERNEST BALDWIN allowing an arginase ” preparation of Aspergillus niger to act upon tetramethylene- diguaidine (which is not attacked by mammalian arginase), while Sendju (1925) found approximately the expected amounts of ornithine and urea when A. oryzae was allowed to act upon arginine.

The evidence for. the presence of arginase in bacteria is not at all conclusive. Ackermann (1909) found optically inactive ornithine among the products of putrefactive decomposition of d-arginine carbonate, while Hino (I 924) found that ammonia was produced when cultures of B. pyocyaneus and B. fluorescens were incubated with arginine, and attributed this to the simultaneous action of arginase and urease. Bacteria killed with acetone showed an argininolytic activity which appeared to extend to 1- as well as to d-arginine, but no arginase could be detected in the culture medium, nor in Streptococcus, Staphylococcus, B. coli communis, B. dysenteriae shiga, B. typhi, B. paratypki and B. prodigiosus. Sendju (1925) also obtained negative results with B. coli. Kossel & Curtius (1925) followed up the

NH NH I

0

H,N-&-HN. CH,. ma. cH, . CHI. NH-&-NHI

H 1 N J - H N . ma. CHI. CHI. ma. NH-&-NHa

NH ‘\, I

\

1 \ ?

\ NH /

0

0

NH-k-NHa HaN. CHI. CHI. CHI. CHI. NH-&-NHS

CHI. CHI. NH-&-NHt ’0 ?

0 O / ,’

0 0

\ \r

\ 0 J. /

\ Y

H,N . CHI . CHI. CHI. CHI. NH, ’

Fig. I . Putrefactive decomposition of arcain, after Linneweh-see text.

work of Hino, but only two of their results could possibly be regarded as definite evidence in favour of the presence of arginase, and these were both obtained with acetone preparations of one strain of B. pyocyaneus. Two other strains gave negative results, and the activity even of this one strain had disappeared after 3 months. Nevertheless, living bacteria of these strains were able to decompose d- and 1-arginine, but as Kossel & Curtius themselves point out, the mere disappearance of arginine cannot be regarded as evidence for the presence of arginase. Hino (1924) had found that arginine may disappear under the action of certain bacteria and that, if it does, ammonia may be produced and the form01 titration value of the solution be increased. But these facts could equally well be explained in another way.

The recent work of Linneweh (1931 a, b, 1932 a, b) is particularly suggestive in this respect. Linneweh studied the breakdown of arcain under the influence of mixed cultures of putrefactive micro-organisms and found that it proceeds according to the scheme of Fig. I. The final products are putrescine, ammonia, carbon dioxide and perhaps traces of urea (dotted lines in the diagram), while agmatine and a series of carbaminyl compounds were isolated as intermediates.

A rginase 259 Such changes as these were already known; thus agmatine was found by Reinwein & Kochinski (1924) to be convertible into putrescine under the influence of putrefactive micro-organisms, while creatine is converted into methylhydantoin (Ackermann, 1913 a, b ; cf. Ellinger & Matsuoka, 1914) under similar conditions. Ackermann (193 I ) has also shown that arginine can be converted into citrullin in this manner and proposes the name “ argininodesimidase ” for the responsible enzyme, but Linneweh (1932 b) suggests the more general term “guanidinodesimidase ” for enzymes which convert the guanidine into the ureido group. Horn (1933)~ working with B. pyocyaneus, obtained 4-9 per cent. of citrullin from arginine, but could obtain none when B. coli was used, and these results compare interestingly with those of the earlier work which could probably be accounted for by guanidinodesimidase. We must suppose that many bacteria contain such an enzyme or group of enzymes. Even in the work of Linneweh the amounts of urea found were minute, and it seems, on the whole, that the existence of a bacterial arginase is an unnecessary postulate.

In conclusion it might be mentioned that Wada (1932) has prepared citrullin from casein by tryptic digestion but could find no reason for supposing that trypsin contains guanidinodesimidase, while Ackermann (1932) finds that the enzyme is probably entirely absent from mammalian tissues.

111. THE FUNCTIONAL IMPORTANCE OF ARGINASE

(I) In ureotelic organisms

It is to Clementi (1914b, 1915a) that we owe the first statement of the generalisation that arginase is associated with ureotelic metabolism. Ureotelic vertebrates excrete the ammonia which arises from the catabolism of proteins mainly in the form of urea. According to classical theory ammonium carbonate is first formed, undergoing two successive dehydrations to yield ammonium carbamate and then urea, but, while there has never been much doubt that urea is formed in the animal organism at the expense of ammonia and carbon dioxide, there has never been much evidence for the occurrence of ammonium carbamate as an intermediary. The chief alternative view has been that supported largely by Fearon (I 926) ; here cyanic acid and ammonium cyanate were regarded as the intermediates, followed by tautomerisation of the cyanate to give urea as in Wohler’s classical synthesis. Here again the evidence is slight.

Ever since the discovery of arginase in 1904 it had been supposed that a part of the urea excreted by the mammals could be accounted for by the action of the hepatic arginase upon ingested arginine, but Krebs & Henseleit (1932) showed that the whole of the urea must derive from the action of this enzyme. The synthesis takes place in a cyclical manner, ornithine acting as a catalyst, in accordance with the scheme of Fig. 2. There is little need to go into any detailed discussion of the evidence for this scheme since the paper is already classical, but the reader may be referred to Krebs’ review (1934) for further information.

The work of Manderscheid (1933) shows that the same cycle operates not only BR X I

a

260 ERNEST BALDWIN in mammals but also in the livers of Amphibia (frog) and of chelonian reptiles (Testudo graecu) and even in the liver of the human foetus at 3-4 months, but not in avian liver. A logical basis has thus been established for the empirical rule enunciated by Clementi, which, as was shown in the first section of this article, applies without serious limitation to the vertebrate phylum, the ureotelic nature of the metabolism of chelonian reptiles (Testudo and Emys) having recently been confirmed by feeding experiments (Clementi, 1929~) .

It is unfortunate that the fishes have not yet been at all thoroughly studied from this new viewpoint. Manderscheid (1933) has examined two fresh-water species (teleosts) and found no evidence for the presence therein of the ornithine cycle, but the results of Hunter & Dauphinee (1g24b) and of Hunter (1929) show that the arginase content of teleostean fishes varies within wide limits. The elasmobranchs have not been examined at all from the new point of view, but they present a case of unusual interest and deserve brief mention here. Krukenberg (1888)

ornithine

' y (arginase) ) a rgin i ne citrulli n

Fig. 2. Cyclical synthesis of urea in mammalian liver (simplified from Manderscheid, 1933).

showed that while urea is absent from members of the Cyclostomata, Cephalo- chordata and Teleostei it is abundantly present in the Elasmobranchii. This observation has repeatedly been confirmed ; thus Kisch (1930) who, incidentally, gives an excellent summary of the earlier literature, found 1.362.54 per cent. of urea in the bloods of ten different elasmobranch species, while entirely similar values were found for the cerebrospinal, pericardial and perivisceral fluids, bile and ear lymph, as well as for the muscles and, in some species, the electrical organs. Urea is essential, moreover, for the maintenance of normal cardiac function in the elasmobranchs (Baglioni, 1906; Mines, 1912; Simpson & Ogden, 1932).

Like the marine teleosts, the elasmobranchs live in an environment the salinity of which is greater than their own, and they are therefore under the necessity' of maintaining a constant internal salinity against an unfavourable osmotic gradient. The teleosts do this (see Smith, 1932) by continually swallowing sea water and actively excreting the unwanted salts, largely by means of special cells situated in the gill membranes. The elasmobranchs, however, meet the situation by raising the total osmotic pressure of the internal above that of the external environment by maintaining in it a concentration of about 2 per cent. of urea, to which the gills are practically impermeable. In the elasmobranchs, therefore, urea is not to be

A rginase 261 considered simply as a metabolic end-product but as a highly soluble, non-toxic crystalloid upon which these fishes rely for the regulation of their internal environment. Correlated with this abundance of urea and the urgency of maintaining it we find that arginase is present in practically every organ of the body, often h high concentration. Kisch (1930), working on dehepatised elasmobranchs, came to the conclusion that the urea is by no means exclusively hepatic in origin, and this seems good reason for supposing that arginase, which is not here confined to the liver, is once more concerned with the production of urea, probably through the ornithine cycle. The results of Sendju (1925) suggest that a similar non-localised synthesis of urea may take place in chelonian reptiles.

Probably the Holocephali too would repay further study. We have already mentioned that Hunter (1929) found Chimaera colliez' to be very poor in arginase, but Krukenberg (1888) states that he was able to isolate urea from the tissues of C. monstrosu. Further investigations would be interesting if only by reconciling these apparently contradictory findings, but it is likely that they would also provide new evidence respecting the much discussed ancestry of the Holocephali.

(2) In uricotelic organisms We have noticed that the livers of birds and reptiles (excluding the Chelonia)

are relatively poor in arginase. The reptiles have not been much studied, but it seems likely from the results of Manderscheid (1933) that the ornithine cycle is absent from the adult hen and also, according to Needham, Brachet & Brown (1935), from the embryo. The urea excretion of adult hens has been carefully studied by Clementi (1932 u, b), who finds that the output on a normal diet averages 70 mg. per day, falling to 20 mg. per day in starvation. The only substance which increases the output, apart from urea itself, is arginine, so that we must attribute the urea excreted by the bird to the action of arginase upon ingested arginine. Needham, Brachet & Brown (1935) have reached the same conclusion for the chick embryo. These results finally exclude the possibility suggested by Wiener (1902) that the formation of uric acid in the bird proceeds by way of urea. In the past a certain amount of evidence has been adduced in favour of Wiener's hypothesis, but in more recent times only negative results have been obtained as, for example, by Russo & Cuscuna (1931) and Cuscuna (1934) with the perfusion technique, and by Benzinger & Krebs (1933) using the tissue slice method. Nor is there any indication in the results of Schuler & Reindel (1933) that urea is in any way concerned in the production of uric acid. The subject has also been exhaustively studied by workers of the Italian school, but again with negative results. Thus urea has been given to birds (hens, geese, pigeons) by injection, the following substances being orally administered at the same time :

Malonic, tartronic and lactic acids (Clementi, 1929b). Pyruvic and propionic acids (Torrisi & Torrisi, 1931, 1932). Dialuric and barbituric acids (Torrisi, 1932, 1933). Glycerol and glyceric acid (Biondi, 193 I). Tartronic, lactic, barbituric and dialuric acids (Russo, 1933).

262 ERNEST B A L D W I N In no case was there any significant increase in the amounts of uric acid excreted,

and in some cases the urea was quantitatively recovered. Minkowski (1886) was the first to demonstrate that ammonium carbonate is converted into uric acid in the avian organism and this remains the only substance known to yield uric acid directly, whether in the adult bird (Clementi, 193oa, 1 9 3 2 ~ ) or in the embryo (Needham, Brachet & Brown, 1935). Whatever the mechanism of uric acid synthesis in the bird may be it is at least certain that urea does not enter into the scheme and that arginase is not concerned in the synthesis.

But it has long been known that birds excrete benzoic acid, which IS for them a dietary constituent of frequent occurrence, in the form of ornithuric acid, i.e. dibenzoylornithine (see Sherwin, 1922 and Lusk, 1928, in this connection), and Clementi (19146, 1915a) pointed out that the ability to effect this synthesis must probably be associated with the presence of arginase in the avian kidney. Crowdle & Sherwin (1923) find that although hens can synthesise ornithuric acid on a protein-free diet, the only aminoacid capable of increasing the output of ornithine is arginine. Evidently, then, although arginase is probably of no importance in the main processes of nitrogenous metabolism in the bird, it is important for the production of ornithine for protective synthesis. It probably accounts also for all the urea excreted on a normal diet.

Of other uricotelic organisms we know little. The literature contains no information regarding the Insecta, but the gastropod molluscs have recently been studied a good deal from this point of view. The snail, Helix pomatia, was shown by Clementi (1918) to contain arginase, and it seemed to Baldwin & Needham (1934) more than coincidence that this animal was also practically the only invertebrate known to excrete significant quantities of urea (Delaunay, 1927, 1931). They confirmed Clementi’s finding of arginase in the hepatopancreas and found it in the nephridium also, but were unable to demonstrate any synthesis of urea by surviving slices of the “liver”. They were led to conclude that Helix resembles the bird, though the possibility of a synthesis could not be finally excluded. At this time, however, there existed no reliable numerical data as to the concentration of arginase in the hepatopancreas, and the subsequent investigations of Baldwin (1935~) reopened the question of a possible synthesis of urea, for it then appeared that the hepatopancreas of Helix contains as much arginase as the most active of the mammalian livers. The failure of Baldwin & Needham (1934) to demonstrate such a synthesis would be explicable if the urea were being transformed into bome other substance as rapidly as formed.

Now there exist among gastropods remarkable correlations between habitat and the production of uric acid (Needham, 1935) and again between uric acid production and arginase content (Baldwin, 1935~) . This suggests that arginase may be in some way concerned in the synthesis of the uric acid which is so characteristic a feature of the nitrogenous excretion of the terrestrial gastropods. It seems possible that urea is first formed, possibly through the ornithine cycle, and then converted into uric acid, perhaps by the mechanism suggested by Wiener (1902), i.e. by condensation with tartronic acid. We have seen that Wiener’s scheme is

A rginase 263 quite inadmissible for the case of the birds, but some experiments by Baldwin & Needham (1934) have shown that the production of uric acid probably follows entirely different paths in the snail and in the bird; furthermore, Baldwin (19353) finds that the snail is able to convert urea and tartronic acid together into uric acid. It has been shown by Howes & Wells (1934) that the snail undergoes alternate periods of high and low water content, and that activity is always associated with a high degree of hydration. Baldwin suggests that in Helix, at any rate, there may also be an alternation of ureotelic and uricotelic types of excretion ; when the water content is high urea may be excreted, but during periods of desiccation this urea, which cannot now be excreted, may be converted into uric acid and stored in that form to be evacuated later.

These speculations have received a certain amount of experimental support 01

the lines just indicated and, so far at least, do not appear to be contradicted by any of the known evidence. They would suggest that unlike the bird, which has developed entirely new mechanisms for producing the uric acid required by a cleidoic development and a terrestrial existence, the snail has modified a previously existing mechanism, urea being formed as the primary end-product of nitrogenous catabolism but being secondarily converted into uric acid. Thus although xerophilous vertebrates and gastropods alike are mainly uricotelic, convergent evolution, in the chemical sense, would appear not to imply the development of identical or even similar chemical mechanisms.

(3) Ingrowth Edlbacher (1917) detected arginase in foetal human liver as early as the fourth

month, and Fuchs (1921) too has found it in the human foetus. According to Manderscheid (1933) the ornithine cycle is already present in the human foetus at the third or fourth month. Hunter & Dauphinee (19243) found that, although absent from the unfertilised elasmobranch egg, the enzyme is abundantly present shortly before hatching when the embryo, according to Needham & Needham (1930), shows a very marked uraemia. Similarly Takahashi (1928) found that the conjugation of benzoic acid with ornithine can be accomplished as early as the ninth day by the chick embryo. Arginase, then, can discharge its adult function quite early in embryonic life. But it is present even earlier than this. Thus in the chick “the capacity to produce ornithine from arginine appears to be present considerably earlier than the capacity to conjugate it to form ornithuric acid” (Needham, Brachet & Brown, 1935). Edlbacher & Mertz (1927) again have detected arginase even in undifferentiated guinea-pig embryos. The latter authors also studied a variety of neoplasmic growths and wrote: “wir gelangen zu dem Schluss, dass wir in dem Argininabbau . . . einen spezifischen Wachstumsfaktor zu erblicken liaben, durch den sich auch das Gewebe maligner Saugertumoren von normalem Gewebe chemisch differenzieren lasst .” If this is true we should expect to find that the arginase content of embryonic tissues progressively falls off as time goes on and the growth rate diminishes and Needham & Brachet (1935) have shown that this is actually the case in the chick. They give the following

264 ERNEST BALDWIN average values for the arginase content of the embryo, the figure at hatching being calculated from the data of Chaudhuri (1927) :

ah 3

$+ I2

Hatched chick

1.16 0.87 0.63 0.09 0.04 0.05

It is known, however, that the glutathione content (Murray, 1926) and the total reducing power (-SH plus ascorbic acid plus unknown reducing substances ; Bierich & Rosenbohm, 1935) of the chick fall off as development proceeds, and it is also known that glutathione, cysteine and ascorbic acid will activate arginase under certain conditions. The observed diminution in arginase activity might therefore be due to the decrease in the amount of activators available, but no increase in activity was observable when glutathione, cysteine and ascorbic acid were added to embryos at 49 and 8 days and to the fifth-day yolk-sac. These results lend valuable support to the theory of Edlbacher which, hitherto, had rested almost entirely upon work with tumour tissue ; but they are also interesting when compared with certain results of Waldschmidt-Leitz & McDonald (1933). According to these authors, although necrotic tissue contains more arginase than the actively growing parts, the arginase activity of a tumour does not increase much as the tumour ages and the proportion of necrotic tissue increases, and this, as the results of Edlbacher, Koller & Becker (1934) show, is correlated with the presence of only very small amounts of reducing substances in the necrotic parts.

Fuchs (1921) used the technique of formol titration to study various pathological conditions including carcinoma of the liver, and found that the metastases contain arginase. Fujiwara (1929) has studied the effect on the arginase content of the host of implanting transplantable tumours in mice, Within 24 hours of implantation the average arginase content of the kidneys of a series of female mice had fallen from 312 to 65 units (Edlbacher’s A.E.) and that of the livers from 4000 to 1400. While it is striking that there is such a profound effect upon the tissues of the host, the injection of arginine alone is said (Edlbacher & Schuler, 1932) to lead to a fourfold increase in the arginase content of the muscles and kidneys of normal guinea-pigs. It seems that these changes must be connected with general rather than specific metabolic processes.

Edlbacher & Mertz (1927) paid special attention to the activity of tumour tissues itself, and found that neoplasmic tissues in general are characterised by a high argininolytic activity. It was upon these observations in the first place that Edlbacher based the generalisation that growing tissues as a whole possess a high arginase activity. In a series of later papers (Edlbacher & Kutscher, 1931, 1932; Edlbacher, Koller & Becker, 1934; Edlbacher & Jung, 1934; Edlbacher, 1934) Edlbacher and his collaborators have elaborated the view that arginase, nuclease and phosphatase are peculiarly associated with a high rate of cell division and

Arginase 265 growth. It is held that arginase is not here concerned with the formation of urea, and this is upheld by the findings of Neber (cited in Edlbacher, Koller & Becker, xyj4), but with the synthesis of the protein components of nucleoplasmic material, probably in connection with the large amounts of arginine which characterise the protamines and histones. That such a correlation between growth and high arginase content exists cannot be doubted, and it is probably significant in this connection that the testis, in which organ alone active cell division persists into adult life, contains more arginase than any other tissue, apart from the liver and the kidney, and, furthermore, that its arginase content increases suddenly at puberty (Edlbacher & Bonem, 1925).

IV. SUMMARY

I . Arginase is a highly specific intracellular enzyme and requires that both the carboxyl and the guanidine groups shall be free if the substrate molecule is to be activated. The kinetics of arginase and its behaviour towards activators and inhibitors are very briefly discussed in the text.

2. Arginase is abundantly present in the liver of ureotelic vertebrates; small amounts are present also in the kidney and testis, but little or none elsewhere. In uricotelic vertebrates it is mainly confined to the kidney.

3. Male animals always contain about 50 per cent. more arginase than females ; a sudden increase in the arginase content of the testis at puberty suggests a possible specific relation between sex and the distribution of arginase.

4. Arginase is widely distributed among invertebrates, usually in small amounts. But the terrestrial gastropods contain as much of the enzyme as do the ureotelic vertebrates.

5 . The synthesis of urea by ureotelic vertebrates takes place by a cyclical mechanism involving arginase. This system is present in mammals, chelonian reptiles and Amphibia, but not in birds. It is very probably present in the elasmobranch fishes also, and the possibility that it is present in the teleosts remains open.

6. Although arginase is not concerned in the production of uric acid by uricotelic vertebrates it does account for such urea as is excreted by these forms. Its main function in the birds is that of supplying ornithine for detoxication.

7. Many invertebrates resemble the birds in possessing small amounts of arginase, which probably suffice to account for the urea which they excrete. In the terrestrial gastropods it is possible that metabolism is primarily ureotelic as in the mammals, but that urea is secondarily converted into uric acid as an adaptation to xerophilous life.

8. Although many bacteria are capable of breaking down arginine they probably contain no arginase but only guanidinodesimidase. This enzyme appears to be absent from vertebrate tissues.

9. Arginase is present early in embryonic life and is soon capable of discharging its adult function. But there exists a correlation between a high growth rate and a high content of arginase, phosphatase and nuclease, which suggests a specific association between arginase and the processes of growth.

266 ERNEST BALDWIN REFERENCES

ABDERHALDEN, E. & SicmL, H. (1929). Fennentforschung, 10, 188. ACKERMANN, D. (1909). Hoppe-Seyl. Z. 60, 482. - (1913~). Z . Biol. 62, 208. - (1913b). Z. Biol. 63, 78. - (1931). Hoppe-Seyl. Z. 203, 66. - (1932). Hoppe-Seyl. Z. 209, 12.

ARVOLD, A. & LUCK, J. M. (1933). J. biol. Chem. 99, 677. BAGLIONI, S. (1906). Zbl. Physiol. 19, 385. BALDWIN, E. (1933). Biol. Rev. 8, 74. - (1934). Biochem. J. 28, 1'55. - -- (1935a). Biochem. J . 29, 7.52. - (1935 b). Biochem. J. (in the Press). BALDWIN, E. & NEEDHAM, D. M. (1933). J. Physiol. 80, 221.

BENZINGER, T. & KREBS, H. A. (1933). Klin. Wschr. 12, 1206. BIERICH, R. & ROSENBOHM, A. (193s). Hoppe-Seyl. 2. 231, 47. BIONDI, G. R. (1931). Arch. Fisiol. 30, 174. BRIDGE, T. W. (1904). In Camb. Nat. Hist. 7, 467 (London). CALWRY, H. 0. & BLOCK, W. D. (1934). 3. 6ioI. Chem. 107, 155. CHAUDHURI, A. S. (1927). J. exp. Biol. 5, 97. CLEMENTI, A. (1914U). R.C. Accad. Lincei (s), 23, 517. - (19146). R.C. Accad. Lincei (5 ) , 23, 612. - (19x5~). Arch. Fisiol. 13, 189. - (19x56). R.C. Accad. Lincei (s), 24, I. - (1916). Arch. Fisiol. 14, 207. - (1918). R.C. Accad. Lincei ( 5 ) , 27, 299. - (1922). R.C. Accad. Lincei (s), 31, 454. - (1929U). R.C. Accad. Lincei (6), 9, 505 . - (1929b). Boll. SOC. ital. Biol. sper. 4, 503 and 1062. - (1930U). Arch. Sci. biol., Napoli, 14, I. - (193ob). Boll. SOC. ital. Biol. sper. 5 , 1142. - (1932~). Arch. ital. Biol. 86, 70. - (1932b). Proc. XIV Congr. int. Fisiol., Roma, p. 24.

CROWDLE, J. H. & SHERWIN, C. P. (1923). J. biol. Chem. 55, 365. CUSCUNA, C. (1934). Arch. Fisiol. 33, 402. DAKIN, H. D. (1903). J . Physiol. 30, 84. - (1907). J. biol. Chem. 3, 435. DELAUNAY, H. (1927). Trau. Lab. SOC. sc i . Arcachon, 1. - (1931). Biol. Rev. 6, 265. EDLBACHER, S. (19x5). Hoppe-Seyl. Z. 95, 81. - (19x7). Hoppe-Seyl. Z. 100, 1 x 1 . - (1934). Rektoratsrede, Base1 Univ.

EDLBACHER, S. & BONEM, P. (1925). Hoppe-Seyl. Z. 145, 69. EDLBACHER, S. & BURCHARD, H. (1931). Hoppe-Seyl. Z. 194, 69. EDLBACHER, S. & JUNG, A. (1934). Hoppe-Seyl. 2. 227, 114. EDLBACHER, S. & KUTSCHER, W. (1931). Hoppe-Seyl. 2. 199, zoo. -- (1932). Hoppe-Seyl. Z. 207, I. EDLBACHER, S. & MERTZ, K. W. (1927). Hoppe-Seyl. 2.171, 252. EDLBACHER, S. & ROTHLER, H. (1925~). Hoppe-Seyl. Z . 148, 264. -- (1925 b). Hoppe-Seyl. Z . 148, 273.

EDLBACHER, S. & SCHULER, B. (1932). Hoppe-Seyl. Z. 206, 78. EDLBACHER, S., KOLLER, F. & BECKER, M. (1934). Hoppe-Seyl. 2.227, 99. EDLBACHER, S., KRAUS, J. & LEUTHARDT, F. (1933). Hoppe-Seyl. 2. 217, 89. ELLINGER, A. & MATSUOKA, 2. (1914). Hoppe-Seyl. Z. 89, 441. ELLIS, A. E. (1926). British Snaih, Oxford. FEARON, W. R. (1926). Physiol. Rev. 6, 399. FELIX, K. & MORINAKA, K. (1923). Hoppe-Seyl. Z. 132, 152. FELIX, K. & TOMITA, M. (1923). Hoppe-Seyl. Z. 128, 40. FUCHS, B. (1921). Hoppe-Seyl. Z. 114, 101. FUJIWARA, H. (1929). Hoppe-Seyl. 2. 185, I. GROSS, R. E. (1920). Hoppe-Seyl. Z. 112, 236. HINO, S. (1924). Hoppe-Seyl. Z. 133, 100.

BALDWIN, E. & NEEDHAM, J. (1934). Biochem. J. 28, 1372.

Arginuse HINO, S. (1926). J . Biochem., Tokyo, 6, 335. HORN, F. (1933). Hoppe-Seyl. Z. 216, 244. H o w , N. H. &WELLS, G. P. (1934). J. exp. Biol. 11, 327. HUNTER, A. (1928). Creatine and Creatinine, London. - (1929). J. biol. Chem. 81, 505 . - (1934). Quart. J. exp. Physiol. 24, 177.

HUNTER, A. & DAUPHINEE, J. A. (1924~). Proc. roy. SOC. B, 97, 209. -__ (19246). Proc. roy. SOC. B, 97, 227. -~ (1930~). Biochem. J. 24, 1128. -- (193ob). J. biol. Chem. 85, 627. -- (1933). Quart. J. exp. Physiol. 23, 119.

HUNTER, A. & MORRELL, J. A. (1922). Trans. roy. S O ~ . Can. 16, v, 75. -- (1914). J . Soc. ehem. Ind., Lond., 43, 691. -- (1933). Quart. J. exp. Physiol. 23, 89.

KARRER, P. & ZEHENDER, F. (1934). Helv. chim. Acta, 17, 737. KIESEL, A. (1922). Hoppe-Seyl. Z. 118, 284. KISCH, B. (1930). Biochem. 2. 226, 197. KLEIN, G. & ZIESE, W. (1932~). Hoppe-Seyl. Z. 211, 23. -- (19326). Hoppe-Seyl. Z. 213, 201. -- (1932~). Hoppe-Seyl. 2. 213, 217. -- (1933). Hoppe-Seyl. 2. 222, 187. -- (1935). Klin. Wschr. 14, 205.

KOSSEL, A. & CURTIUS, F. (1925). Hoppe-Seyl. Z. 148, 283. KOSSEL, A. & DAKIN, H. D. (1904~). Hoppe-Seyl. Z. 41, 321. -- (1904b). Hoppe-Seyl. Z. 42, 181.

KREBS, H. A. (1934). Ergebn. Enzymforsch. 3, 247. KREBS, H. A. & HENSELEIT, K. (1932). Hopfie-Seyl. Z. 210, 33. KRUKENBERG, C. F. W. (1888). Ann. Mus. Hist. nut. Marseille, 3, No. 3. KUTSCHER, F. & ACKERMANN, D. (1933). Ann. Reu. Biochm. 2, 355. KUTSCHER, F. & SEEMANN, J. (1901). Hoppe-Seyl. Z. 34, 528. -- (1902). Hoppe-Seyl. 2. 35, 432. LEUTHARDT, F. & KOLLER, F. (1934). Helv. chim. Acta, 17, 1030. LIEBEN, F. & LIEBER, H. (1934). Biochem. 2. 275, 38. LINNEWEH, F. (1931a). Hoppe-Seyl. Z. 200, 1x5. - (1931b). Hoppe-Seyl. Z. 202, I. - (1932~). Hoppe-Seyl. Z. 205, 126. - (1932b). Hoppe-Seyl. Z. 207, 152.

LUSK, G. (1928). The Science of Nutrition, 4th ed. London & Philadelphia. MANDERSCHEID, H. (1933). Biochem. Z. 263, 245. MEYERHOF, 0. (1928). Arch. Sci. biol., Napoli, 12, 536. MEYERHOF, 0. & LOHMANN, K. (1928). Biochem. Z. 196, 54. MIHARA, S. (1911). Hoppe-Seyl. 2. 76, 443. MINES, G. R. (1912). J. Physiol. 43, 467. MINKOWSKI, 0. (1886). Arch. exp. Path. P h n n a k . 21, 41. MOCHIZUKI, J. & KOTAKE, Y. (1904). Hoppe-Seyl. 2. 43, 165. MURRAY, H. A. (1926). J. gen. Physiol. 9, 621. NEEDHAM, J. (1935). Biochem. J . 29, 238. NEEDHAM, J. & BRACHET, J. (1935). C.R. SOC. Biol., Paris (in the Press). NEEDHAM, J., BXACHET, J. & BROWN, R. K. (1935). J. exp. Biol. (in the Press). NEEDHAM, J. & NEEDHAM, D. M. (1930). J. exp. Biol. 7, 7. NORTHROP, J. H. (1920). J. gen. Physiol. 2, 471. PURR, A. & WEIL, L. (1934). Biochem. 3. 28, 740. REINWEIN, H. & KOCHINSKI, K. L. (1924). Z. BWl. 81, 291. RICHET, C. (1894). C.R. Soc. Biol., Paris, 46, 525. - (1897). C.R. SOC. Biol., Pans, 49, 743.

RIESSER, 0. (1906). Hoppe-Seyl. Z. 49, 210. Russo, G. (1933). Arch. Fisiol. 33, 124. Russo, G. & CUSCUNA, C. (1931). Boll. SOC. ital. Biol. sper. 6, 250. SALASKIN, S. & SOLOWJEW, L. (1931). Hoppe-Seyl. 2. 200, 259. -- (1932~). Hoppe-Seyl. Z. 208, 171.

SALASKIN, S., SOLOWJEW, L. & TJUKOW, D. (1932). Hopfie-Seyl. Z. 206, I . SCHULER, W. & REINDEL, W. (1933). Hoppe-Seyl. 2. 221, 209 & 232. SENDJU, Y. (1925). J. Biochem., Tokyo, 5, 229.

'

-- (1932b). Biochem. Z. 250, 503.

268 ERNEST BALDWIN SHERWIN, C. P. (1922). Physiol. Rev. 2, 238. SHIGA, K. (1904). Hoppe-Seyl. Z . 42, 502. SIMPSON, W. W. & OCDEN, E. (1932). J. exp. Biol. 9, I . SMITH, H. W. (1932). Quart. Rev. Biol. 7 , I . SOLOWJEW, L. & MARDASCHEW, s. (1932). Hoppe-Seyl. z. 209, 239. S ~ E I B , H. (1926). Hoppe-Seyl. 2. 155, 279 & 292. TAKAHASHI, M. (1928). Hoppe-Seyl. Z . 178, 294. THOMAS, K. (1913). Hoppe-Seyl. Z . 88,465. THOMAS, K., KAPFHAMMER, J . t & FLASCHBNTRXDBR, B. (1923). Hoppe-Seyl. Z . 124, 75. TORRISI, D. (1932). Boll. SOC. ital. Biol. sper. 7, 42. - (1933). Arch. Fisiol. 32, 87. TORRISI, D. & TORRISI, F. ( ~ 9 3 1 ) . Boll. SOC. ital. Biol. sper. 6 , 262. -- (1932). Arch. Sn'. biol., Napoli, 16, 589.

WADA, M. (1932). Biochem. Z . 257, I . WALDSCHMIDT-LEITZ, E. & MCDONALD, E. (1933). Hoppe-Seyl. Z . 219, 115. WALDSCHMIDT-LEITZ, E., SCHARIKOVA, A. & ScHxPpNm, A. (1933). Hoppe-Seyl. Z . 214, 75. WALDSCHMIDT-LEITZ, E., WEIL, L. & PURR, A. (1933). Hoppe-Seyl. Z. 215, 64. WEIL, L. & RUSSELL, M. A. (1934). J. biol. Chem. 106, 505 . WIENER, H. (1902). Beitr. chem. Physiol. Path. 2, 42.

ARGINASE

Documents

Transcript of ARGINASE