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Arginine has been estimated by precipitation as the picrate and flavianate complexes, enzymically by the action of arginase followed by the estimation of the urea formed, microbiologically and colorimetrically (cf. Block & Boiling, 1947). The colorimetric method developed by Sakaguchi (1925a, b) has been most widely used. This method relies upon the formation of a red-coloured complex produced by arginine and other monosubstituted guanido compounds in the presence of a-naphthol and alkaline hypobromite or hypochlorite. Although this method will qualitatively detect small quantities of arginine, it is unreliable quantitatively, as evidenced by the large number of modifications that have been suggested, and which have been reviewed recently by G6mez & Marenzi (1953). A colorimetric method for the estimation of both mono-and di-substituted guanidines was introduced by Eggleton, Elsden & Gough (1943). These compounds give a red-coloured complex with diacetyl and a-naphthol in alkaline solution. The authors found that monosubstituted guanidines such as arginine and glycocyamine produced only one-ninth of the colour given by asymmetrically disubstituted guanidines such as creatine. This observation has been confirmed by Ennor & Stocken (1953) and by Roche, Thoai & Hatt (1954). Although this method is less sensitive than the Sakaguchi method, it is simpler and more reliable. This paper describes a modification of the above reaction. When the colour is developed in the presence of n-propanol and an increased concentration of x-naphthol, the sensitivity of the method for monosubstituted guanidines is equal to that previously obtained with disubstituted guanidines. Thus it is possible to estimate arginine in amounts ranging from 10 to 100 ,ug. EXPERIMENTAL Materials n-Propanol. n-Propanol was purified by distillation after refluxing over NaOH and Al powder. a-Naphthol. The commercial material was light brown in colour and was purified by two steam-distillations from 2N-H2SO4. It was then dried in vacuo at 60°, and dissolved in n-propanol to give a 25 % (w/v) solution, and stored in a dark bottle at-20°. Stock diacetyl solution. A stock diacetyl solution (about 1 %) was prepared from dimethylglyoxime as described by Walpole (1911). Arginine. Arginine hydrochloride was recrystallized from 0-4N-HC1 (cf. Greenberg, 1951), and its purity was checked by N analysis. Other guanido compounds. Guanidine (A.R.) was used. Creatine was recrystallized as described by Hunter (1928). Glycocyamine (guanidoacetic acid) was recrystallized from water. N-Ethylguanidoacetic acid (negmine) was kindly supplied by Dr M. D. Armstrong (see Ennor, Rosenberg & Armstrong, 1955). a-Guanidopropionic acid, ,-guanidopropionic acid and ,-guanidoethylsulphonic acid (taurocyamine) were prepared by guanylation with S-ethylthiourea of alanine, ,-alanine and taurine respectively, as described by Brand & Brand (1942) for the preparation of glycocyamine. 1:2-Diguanidoethane, 1-guanidoethane and 2-guanidoethanol were prepared from ethylenediamine, ethylamine and et...

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FORMATION OF AMMONIA FROM GLUTAMINE 143 presence of IMP and CP resulted in about 97 % inhibition of production of ammonia. This result would be expected if the enzyme responsible were the brain glutaminase described by Krebs (1935). With inorganic phosphate (5 or 10 tmoles/ml.) replacing the IMP and CP in the system, addition of glutamic acid (4 /Lmoles/ml.) was accompanied by a very marked decrease in production of ammonia. Greenstein & Leuthardt (1948) found that bicarbonate buffer did not activate glutaminase. Further evidence for this has been found in the low blank values. They measured the stimulatory effect of various ions on glutaminase, and some organic derivatives of phosphate and arsenate. The present study has included more multivalent ions, since Greenstein & Leuthardt (1948) had shown that univalent ions were poor stimulators ofthe reaction. It was concluded that, in general, as the ionic charge increased so did the effectiveness in stimulating production of ammonia (Table 5). The effect obtained by Weil-Malherbe (1953) can be readily explained on the basis of an anion activation of brain glutaminase by phosphate formed by hydrolysis during the incubation, or by phosphate and sulphate added initially in these solutions. SUMMARY 1. The formation of ammonia from glutamine by rat-brain particles in the presence of inosine triphosphate, or inosinic acid and creatine phosphate, is explained by the presence of low concentrations of phosphate and sulphate which activate the glutaminase present. 2. The reaction is 97 % inhibited by the addition of L-glutamic acid. 3. The effect of various other ions on the reaction was studied. The results indicated that, as the ionic charge increases, so does the efficacy in stimulating production of ammonia. 4. The synthesis of adenosine 5'-monophosphate from inosinic acid does not occur in this system under these conditions.

For the study of biological systems involving; phosphate transfer it is desirable to have an aeciirate method for tlie estimation of inorganic and labile phosphates. The method of Piske and Subbarow (1925) has been modified by many authors Init siieh modifications have been pi'imarily concerned \\itii a seareli for conditions under whicli preferential reduction of phosphomolybdic aeid would take place in the presence of molybdic acid. None of these modifications is readily applicable in the presence of acid-molybdate labile compounds such as pliosphocreatine, acetyl phosphate, or ribose phosphate idtliou(iii attempts liave been made to overcome these difficulties by various procedures. An important modification of the ori<;inal Fiske and Subbarow method was introduced by Lowry and Lopez ('lJ)4(j} who used ascorbic acid as a reducint]; agent at pll :j-;'j-4-2 in the presence of a low molybdatc concentration. Tnder these eonditions labile compounds are much more stable than under the conditions used by Fiske and Subbarow (1925). An objection to the method of Lowry and Lopez may l)e raised, however, on the basis of interfering snbstanees since tliese authors state "in certain tissue extraets the reaction is delayed and iin internal standard must be used".Many of the diwadvantajjes inherent in the Fiske and Subbarow method and modifications thereof have been discussed by Berenblum and Chain (1938 a) and these authors (lO.'iSb) introduced a new principle in that tbe phosphomolybdic acid is removed from the aqueous phase by extraction with isobutanol. Interfering substances are thus largely left behind and reduction is carried out lu the aleohol phase and in tbe absence of molybdic acid.It occurred to us that, if the reaction between phosphate and molybdic acid was fast enough and that if tbe resultant phosphoniolybdie acid could be extracted from tbo aqueous pbnse in a few seconds the contribution which a molybdate-labile or^ano phosphate could make to the ti'ue inortianic phosphate would be minimized. It is the purpose of this paper to detail the investigations 1 The subject matter of this paper was presented at a meeting of the Australian and New Zealand Association for the Advanfement of Science in Hobart in January, 1949. •which have been carried ont nn the reactions involved and to describe a rapid method for the determination of inorfjanic pbosphate in tlic presence of certain acid-molybdatc liibilo phosphorus compounds. METHODS.Reagents.(1) Sodium plioaphocrentine-prcpnred fia dpscribed by Ennor and Stocken (1947)-all solutions were mai\o iip in distilled watpv ;ind the pH ndjustrrt to nbniit pH 7*ri hy the addition of NaOH.Measurevient of rtihvr iiitensitie.'i. All measurements were made in 1 em. cella on the Spekker Photoabsorptiometer using Ilford Filter No. 604, Spectrum Red. RESULTS."In the original method na dpficrilied hy Rerpiihlum nnd Cliriiii (]9r!Sli) it ivns recnmincnded that the concentration of HoSOj and animnninm molylid;it<' h? O-fi . V and 1-25 p.c respectively and that the phosphomolybdic ...

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