BIOSYNTHESIS OF UREA

III. FURTHER STUDIES ON ARGININE SYNTHESIS FROM CITRULLINE*

BY S. RATNER AND BARBARA PETRACK

(From the Department of Pharmacolog?J, New York University College of Medicine, New York, New York)

(Received for publication, March 21, 1951)

Previous studies on the enzymatic mechanism of urea synthesis in mam- malian liver have shown that an isolated enzyme system, prepared from acetone-dried liver powders, catalyzes a high energy phosphate-dependent synthesis of arginine from citrulline. On the basis of preliminary evidence, a reaction mechanism was formulated which involves two distinct steps. In the first step (Reaction 1, a), aspartic acid and citrulline, in the presence of Mg” and adenosinetriphosphate (ATP), condense to form an inter- mediary condensation product with the simultaneous formation of inor- ganic phosphate. In the second step (Reaction 1, b), the intermediate is hydrolyzed to form arginine and malic acid (1).

(1, a) L-Citrulline + n-aspartic acid + ATP = intermediate + ADPi + ‘PO,

(1, b) Intermediate + Hz0 = n-arginine + Z-malic acid

The present communication is concerned with the separation of these two enzymes, the assay and partial purification of the condensing enzyme, and the results obtained thus far in studies of the mechanism of each reaction.

Separation of Condensing Enzyme from Hydrolyzing Enzyme-A S-fold purification of the enzyme system catalyzing the over-all conversion of citrulline to arginine (Reactions 1,~ and b) by means of low temperature alcohol fractionation was described in the earlier study. By ammonium sulfate fractionation, the two enzymes catalyzing the postulated partial reactions have now been separated. This is illustrated in Table I. The data were obtained under the same experimental conditions as those de- scribed previously. For purposes of the experiment, each fraction was dialyzed overnight against 0.05 M phosphate buffer, pH 7.5. This led to considerable loss of activity. Nevertheless, while each fraction showed no more than a trace of activity when tested alone, the combination of either one of the first two fractions with either one of the last two resulted in a

* Aided by grants from the American Cancer Society (recommended by the Com- mittee on Growth of the National Research Council), the Office of Naval Research, and the United States Public Health Service.

1 Adenosinediphosphate.

693

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694 BIOSYNTHESIS OF UREA. III

much larger increase in activity than would be expected from mere summa- tion. Since arginine was estimated as urea, an excess of arginase was added should this enzyme become limiting in the course of fractionation. By investigating each fraction separately with respect to the products of the reaction, it became apparent that condensing activity (Enzyme A) was associated with the fractions precipitating at the higher salt concentration, and hydrolyzing activity (Enzyme B) with the lower fractions.

That Enzymes A and B may be entirely freed of each other is shown in the lower section of Table I. Fraction 3C, corresponding to Enzyme A,

TABLE I Separation of Condensing Enzyme from Hydrolyzing Enzyme

In addition to enzyme, each tube contained the following, expressed as micro- moles per 4 ml.: 5 ATP, 20 r,-aspartic acid, 20 L-citrulline, 50 n-3-phosphogiyceric acid, 13 magnesium sulfate, 100 potassium phosphate, pH 7.5, muscle extract 8 mg., arginase 21 units; 38”; 20 minutes. Fraction 2B was obtained by refractionation of Fraction 2, and Fraction 3C by refractionation of Fraction 3.

Fraction No. %x2- saturation

2B 20-30 40.5* 3c 4l350 16.6

per cent w.

o-20 29.6 20-30 20.5 3040 22.5 &50 31.2

1 ?rotein used Fractions combined

+ +

+ +

+

+

+

+

- 6.: Arginine formed,H.. .._..__. 17 1.8 0 4 0 0 7.2

/.I lH.1

+ +

+

+

- 1.6

+

- 0.0 0.t

+

T- -

+ + - 3.6

* Amount used represents a large excess of Enzyme B.

was obtained by refractionation of Fraction 3 and represents the fraction precipitating at 40 to 50 per cent saturation. Fraction 2B (Enzyme B) was obtained by refractionation of Fraction 2 and represents the fraction again precipitating between 20 and 30 per cent saturation. Since arginase was uniformly present, the absence of activity when Fractions 3C and 2B were tested alone indicates complete lack of mutual contamination.

Method of Following Condensation Reaction-A method has not yet been developed for estimating the intermediary condensation product directly and the rate of condensation has therefore been followed by measuring the disappearance of citrulline. Although this has the obvious disadvantage of following a change in substrate concentration, the calorimetric method employed is reasonably rapid and, as shown below, highly specific under the experimental conditions employed.

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S. RATNER AND B. PETRACK 695

The two calorimetric methods described by Archibald for the estimation of urea (2) and citrulline (3), respectively, differ in the choice of diketone employed for color development. With ac-isonitrosopropiophenone, urea can be estimated in the presence of citrulline, since the color formed with urea is about 10 times greater than with citrulline. The latter method was employed in studies of the over-all reaction, an appropriate correction hav- ing been made for the remaining citrulline. However, in the method for citrulline estimation, the diacetyl monoxime employed develops consider- able color with urea. As pointed out by Archibald, the application of this method is therefore limited to materials from which urea is absent or has

TABLE II

Comparison of Condensing Enzyme Assay by Citrulline Disappearance and Urea Formation

Fractions 3A, 3B, and 3C were obtained by refractionating Fraction 3 (Table I). Fraction 4 is the same as in Table I. Enzyme B is Fraction 2B, Table I; 40 mg. were added where indicated. All fractions were dialyzed against 0.05 M potassium phosphate, pH 7.5. Conditions as in Table I. 21 units of arginase were added to the tubes containing Enzyme B.

Specific activity*

Enzyme A Ammonium sulfate saturation

A citrulline Urea form;h&Enzyme B

jer Gent

3A O-30 0.57 0.74 3B 3040 1.83 2.00 3c 40-50 2.50 2.55 4 40-50 2.21 2.45

* Specific activity is defined as micromoles of citrulline disappearance or urea formation per mg. of protein per hour.

been removed. When this procedure is used to follow the activity of En- zyme A by citrulline disappearance, the formation of urea by contaminat- ing hydrolyzing enzyme, in the presence of arginase, will cause appreciable error.

In order to test the validity of this procedure, various Enzyme A frac- tions, obtained by refractionation of Fraction 3 (Table I), were assayed by the A citrulline method and by the urea method. In the latter case, a large excess of Enzyme B and of arginase was added to limiting amounts of Enzyme A. These are compared in Table II, where’it may be seen that the specific activities arrived at by the two methods are in good agreement.

Nature of Hydrolyzing Reaction--With preparations of Enzyme A free of Enzyme B, the intermediary condensation product accumulates and has been isolated as the barium salt (4). It is quite stable and act,s as a spe-

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696 BIOSYNTHESIS OF UREA. III

cific substrate for Enzyme B. As is to be expected from a disubstituted gua.nido derivative, the intermediary product fails to give more than traces of color under the conditions employed for estimating arginine by the Sak- aguchi method (5) or for estimating creatine with diacetyl and cu-naphthol in alkaline solution (6). In the presence of arginase and an excess of En- zyme B, hydrolysis of the intermediate appears to be complete, and ad- vantage has been taken of this behavior in the quantitative estimation of the compound. Its isolation, purification, and characterization will be described in a separate paper.

Investigation of Reaction 1, b, with the isolated intermediate as sub- strate, confirms the formulation of the enzymatic mechanism as being a purely hydrolytic reaction. Neither ATP nor Mg++ is required and the rate of hydrolysis is not influenced by the absence of inorganic phosphate. The time course of the reaction has been investigated by estimating the arginine and malic acid formed as hydrolysis progresses. It may be seen from Table III that the products2 appear in equivalent amounts, regard- less of the fraction of intermediate remaining.

The results indicate that the intermediate is homogeneous within the limits of assessed purity and that, in agreement with the assigned structure, it behaves, enzymatically, as a derivative of arginine and malic acid. Hy- drolytic cleavage must then occur between one of the guanido nitrogen atoms and the carbon atom originally present in the LY position of aspartic acid.

Nature of Condensation Reaction

Since the enzymatic pathway of arginine synthesis from citrulline and aspartic acid appears to be novel in many respects, the system has been studied in detail in an effort to elucidate the fundamental aspects of the participation of high energy phosphate (-ph) in the transfer of amino groups and the synthesis of guanido groups. This has been approached by investigating the stoichiometry of substrate disappearance, by following phosphate transfer, and by seeking evidence of direct phosphorylation.

Relationship of Intermediate Formation to Citrulline and Aspartic Acid- Since the intermediate can be determined by enzymatic hydrolysis, an in- vestigation of the condensation reaction has been carried out which demon- strates that aspartic acid and citrulline are each utilized in amounts which are equivalent to the intermediate formed. These experiments, as shown in Table IV, were conducted in two steps. Incubation mixtures were set up containing an excess of citrulline, varying amounts of aspartic acid, and sufficient condensing enzyme to catalyze the complete utilization of the

* The identification of arginine and malic acid by isolation and by means of highly specific enzymatic methods has already been described (I).

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S. RATNER AND B. PETRACK 697

aspartic acid. A similar series was prepared containing aspartic acid in excess and varied amounts of citrulline. After incubation the reaction was stopped by heat inactivation, and sufficient arginase and hydrolyzing en- zyme were added to split all of the intermediate formed in the first step. The results given in Table IV are in substantial agreement with the formu- lation of Reaction 1, a. They indicate also that side reactions are virtually

TABLE III Progressive Enzymatic Hydrolysis of Intermediary Condensation Product

In addition to the intermediate, each tube contained 2.43 mg. of hydrolyzing enzyme, specific activity 10, 21 units of arginase, 0.3 ml. of M potassium phosphate, pH 7.5, and malate where indicated in a final volume of 3 ml. After incubation at 38”, the tubes were heated at 100” for 2 minutes, the pH brought to 5.0 with 0.08 ml. of 2 N HCl, and centrifuged. Urea was estimated in 0.5 ml. of the filtrates ob- tained by adding 0.5 ml. of the incubation mixture to 4 volumes of 8.3 per cent tri- chloroacetic acid. Malate was estimated in small Warburg vessels of 6 ml. capacity. Each vessel contained 1.0 ml. of supernatant and 0.2 ml. of M potassium phosphate, pH 5, in the main space. The side arm contained 0.05 ml. of MnCL and 20 mg. of lyophilized bacteria (&co2 5000) in 0.25 ml. of water; gas space, nitrogen; tempera- ture, 25”. All values are expressed in micromoles.

A barium-free solution of the intermediate, containing 14.7 PM per ml., was pre- pared by dissolving 40.0 mg. of the barium salt in 4.0 ml. of water, adding 0.4 ml. of 0.5 M potassium sulfate, and centrifuging the mixture. Assuming 1.5 atoms of Ba per mole, the salt was estimated to be 81 per cent pure.

Expe&ynt Additions

Incubation time Arginine formed Malic acid formed

Intermediate Malic acid

min.

1 7.3 0.0 0 0.0 0.0 2 14.7 0.0 15 5.7 6.0 3 14.7 0.0 35 10.2 10.4 4 14.7 0.0 40 14.3 14.5 5 o.o* 15.0 60 0.0 15.1

* 7.3 PM of intermediate were added to this tube only after the incubated mixture had been heat-inactivated. Malate recovery was thus estimated after exposure to hydrolyzing enzyme and in the presence of unhydrolyzed intermediate.

absent and that the disappearance of citrulline measures the extent to which condensation has occurred.

High Energy Phosphate Transfer in Relation to Formation of Intermedi- ate-As previously reported, Reaction 1, a specifically requires high energy phosphate in the form of ATP. The transfer of phosphate from ATP dur- ing formation of the intermediate is evidenced by the appearance of inor- ganic phosphate. When phosphoglyceric acid (PGA), in the presence of catalytic amounts of ATP, serves as the -ph donor, pyruvic acid appears as well. It might be expected, therefore, that phosphorylation of one of

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698 BIOSYNTHESIS OF UREA. III

the amino acid substrates, presumably citrulline, would take place initially, and that inorganic phosphate lvould be split out subsequently during con- densation. Nevertheless, it has not yet been possible to obtain evidence of phosphorylation apart from condensation.

An alternative hypothesis, that phosphorylation occurs simultaneously with condensation, appears to be less likely for the reason that more in- organic phosphate is formed than intermediate. The ratio of the appear- ance of inorganic phosphate to citrulline disappearance varies with different preparations of condensing enzyme and with the experimental conditions,

TABLE IV

Equivalence of Citrulline and Aspartic Acid, Utilization to Amount of

Intermediate Formed

Each tube contained the following, expressed as micromoles per 4 ml.: 200 TAM buffer, pH 7.5, 26 magnesium sulfate, 5 ATP, 50 n-3-phosphoglyceric acid, 0.8 mg. of muscle extract, 11.4 mg. of condensing enzyme, specific activity 5.0; other ad- ditions as indicated. After 40 minutes at 38”, the tubes were heated at 60” for 2 minutes. To 1 ml. samples of each were added 50 PM of TAM buffer, 21 units of arginase, 6.5 mg. of hydrolyzing enzyme, specific activity 10.0; the mixture was in- cubated at 38” for 60 minutes. The arginine formed was then estimated as urea. All values are given in micromoles.

Additions Citrulline disappearing Intermediate formed

I-Citmlline >-Asp&ate

30.7 7.5 7.6 7.8 30.7 11.2 11.1 10.8 30.7 15.0 15.4 14.2

7.5 30 7.5 7.1 11.2 30 11.2 10.9 15.0 30 15.0 14.1

but has always been found to be greater than 1, although less than 2. The ratios obtained in some representative experiments are given in Table V. The values for inorganic phosphate may be considered to be a reliable measure of the detectable transfer of mph, since the amount of pyruvate formed in each case was almost as high. Pyruvate and phosphate forma- tion in the absence of aspartic acid were identical with the values obtained in the absence of citrulline or of both amino acids and for this reason have been ascribed to contaminating ATPase activity and applied as a correction to the values found in the presence of both amino acids.

If it is assumed, despite the present lack of direct evidence, that a pre- liminary phosphorylation does occur and that the phosphorylated amino acid is formed in excess over the rate of condensation, but is very labile or

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S. RATNER AND B. PETRACK 699

is split by a phosphatase, then variable ratios greater than 1 might be ex- pected. The extent of phosphorylation, and consequently the ratio, might then increase with increasing amounts of ATP. This was investigated in the absence of PGA at initial ATP concentrations of 10, 20, and 30 PM.

Ratios of 1.61, 1.77, and 1.98 respectively were obtained. While these increases in ratio may perhaps indicate an increased phosphorylation, they should be regarded with some reservation. At the higher ATP concen-

TABLE V

High Energy Phosphate Transfer in Relation to Citrulline Disappearance and Formation of Intermediate Product

Each tube contained the following, expressed as micromoles per 4 ml.: 200 TAM buffer, pH 7.5, 26 magnesium sulfate, 5 ATP, 50 n-3-phosphoglyceric acid, 30 L-

aspartic acid, and 30 L-citrulline where indicated, 5.5 mg. of condensing enzyme specific activity 8.0, 0.4 mg. of muscle extract;* 20 minutes; 38”. All A values are expressed as micromoles.

Experi- ment NO.

Complete systemt Aspartate omitted Citrulline “ Both omitted Complete systemt Aspartate omitted Complete system,t 10 min.

“ “ 20 “ “ ‘I 30 “

‘ A

:itrulline

-10.0

-11.4

-5.6 -10.0 -14.5

A pyruvate

t13.5 +5.0 +5.2 t5.2

+16.8 +4.4

-

5 A

bhosphatl

+15.s +4.7 +4.7 f4.7

+19.2 +5.6 +s.4

+13.3 +19.8

d : - 1

Ratio

, phosphate !TzLaz

1.58

1.68

1.50 1.33 1.36

* The muscle fraction employed here was subjected to heat treatment to reduce the ATPase activity (see “Experimental”).

t The phosphate and pyruvate values have already been corrected by the values obtained in the absence of amino acid.

trations there is a marked lowering of activity, which introduces a greater error in the measurement of citrulline differences (see Fig. 1).

Condensing Enzyme Activity with ATP As Only -ph Donor-In agree- ment with observations made on the over-all system, the condensation re- action specifically requires wph in the form of ATP. Fig. 1, Curve 2, gives values for the rate of condensing enzyme activity at increasing con- centrations of ATP as the only wph donor. It can be seen that the highest value of 6.3 PM is reached with 14.5 PM of ATP and that a progressive inhibition becomes apparent with larger amounts. The various substrate concentrations used for obtaining the data of Curve 2 represent the most favorable conditions found with ATP alone; yet this optimum is about 50

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700 BIOSYNTHESIS OF UREA. III

per cent of the rate found with the same preparation in the presence of PGA and 5 PM of ATP, represented by the maximum of Curve 1, Fig. 1.

Other experiments show, moreover, that with 15 PM of ATP enzyme activity is not linear with time or with enzyme concentration. The use of ATP alone is thus unsatisfactory and some attempt has been made to in- vestigate the cause.

The diminished activity has been found to be due to at least two factors,

0 4 8 12 16 20 24 28 pM ATP OR AMP

FIG. 1. Condensing enzyme activity in the presence of high concentrations of ATP. Curve 1, with PGA and varying concentrations of ATP; Curve 2, with vary- ing concentrations of ATP; Curve 3, with ATP concentration constant and varying concentrations of AMP. For Curves 2 and 3, the tubes contained, per 4 ml., 20 PM

each of L-aspartic acid and L-citrulline, 26 PM of magnesium sulfate, 200 PM of TAM buffer, pH 7.5, 4.5 mg. of enzyme, specific activity 8.0, and ATP as indicated. For Curve 3, AMP was added as indicated to 14.5 /LM of ATP. For Curve 1, the incuba- tion mixture was similar except that 30 PM of each amino acid were present in addi- tion to 50 PM of n-3-phosphoglyceric acid and 2.4 mg. of muscle extract. Enzyme was added after temperature equilibration at 38”. Incubation time, 20 minutes.

one being an impurity in the ATP and the second factor adenylic acid per se. The ATP effect has been studied separately by using the system con- taining PGA and muscle extract. When the ATP concentration was in- creased beyond the 5 PM level, which gives maximum activity, an increasing inhibition occurred which amounted to 22 per cent at 14.5 PM. This ap- pears to be due to a contaminant of the ATP, for a similar series of experi- ments carried out with adenosinediphosphate (ADP) and adenosinemono- phosphate (AMP) showed practically no inhibition at the same level. Both the ADP and AMP added must have been rapidly phosphorylated by the active phosphorylating system containing PGA and muscle extract, high in myokinase. It is unlikely that the contaminant is a heavy metal,

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S. RATNER AND B. PETRACK 701

although inhibition of this type has been reported from several laboratories (7, 8). Contaminating heavy metals have been removed as rigorously as possible by subjecting all the compounds tested to ion exchange treatment on Amberlite IR-100, as described by Polis and Meyerhof (7). In addition, possible inhibition by Na+ (9, 10) was avoided by employing a K+-charged resin. The source and analytical data pertaining to these compounds are given in the experimental section.

While it appears that when ATP is employed in very high concentrations some contaminant, other than heavy metal, exerts a direct inhibition on the condensation reaction, this has not seemed an entirely satisfactory ex- planation for the reason that the maximum of Curve 2 (Fig. 1) is lower than would be predicted from the data of Curve 1. A second and significant cause of inhibition has been found in AMP. This may be seen in Curve 3, which was obtained by adding increasing amounts of AMP to an incubation mixture containing 14.5 pM of ATP. The inhibition is greater than that caused by an equivalent amount of ATP, and with 7.5 PM it amounts to 35 per cent. Significant inhibition by ADP has been excluded.

Preparations of the condensing enzyme display myokinase activity when assayed spectrophotometrically (11). The presence of this enzyme is also shown by the observation that ADP can act, though poorly, as the ex- clusive wph donor. It is therefore quite likely that AMP is formed during incubation. Assuming myokinase equilibrium (12), it can be calculated from the uncorrected inorganic phosphate formed at the maximum point of Curve 2 that about 7 pM of AMP may be formed.

Adenylic acid inhibition has previously been observed in the glycolysis of brain tissue by Greenberg (13). In the single ATP-requiring reaction investigated in the present studies, the inhibitory effect shown by AMP suggests that the mechanism of inhibition may be one of competition for the site of ATP-enzyme interaction.

Some Properties of Condensing Enzyme-As shown above, the highest enzyme activity was observed in the presence of an excess of PGA, small amounts of ATP, and muscle extract. With the condensing enzyme alone, concentration-dependence curves for PGA, ATP, citrulline, aspartic acid, and Mg++ are about the same as were found for the over-all system. With a modified medium (see “Experimental”), the velocity of. condensation follows a linear course with enzyme concentration and with time up to 30 minutes, provided no more than half the citrulline has been removed. The unit has been defined as the amount of enzyme which forms 1 PM of inter- mediate per hour.

h’W+ exerts a marked protective action which is most evident during temperature equilibration at 38” and is not shown by the amino acid sub- strates. At 38”, enzyme activity falls off rapidly with time, to the extent

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702 BIOSYNTHESIS OF UREA. III

of 33 per cent in 7 minutes, and Mg++ prevents this loss. On the other hand, about 40 to 50 per cent of the activity is lost on dialysis against dilute phosphate or tris(hydroxymethyl)aminomethane (TAM) buffer at 7.5, and Mg++ only partially prevents this loss.

The effect of other metallic ions has been investigated. At 0.001 M, Ni++ and Co++ had negligible effects. Fe+++, Zn++, and Fe++ inhibited to the extent of about 20 per cent, while Mn++ and Ca++ each inhibited to the extent of 56 per cent. No inhibition was shown by cyanide at 0.001 or 0.002 M. The large inhibition by Ca++ and Mn++ is of interest in that it may be due to competition with Mg++ for the enzyme. Although the ef- fect of Ca++ might possibly be explained by activation of ATPase, one would hardly expect so pronounced an inhibition through ATPase alone in an actively phosphorylating system with PGA in large excess. It has been previously noted that fluoride also inhibits. In view of the strong inhibi- tion by Mn++, precautions have been taken to remove, with dilute phos- phate, excess Mn++ from Mn++-activated arginase preparations whenever studies of the over-all reaction required the addition of this enzyme.

Enzyme activity in the presence of glycylglycine or TAM buffer at pH 7.5 is approximately 20 per cent greater than in phosphate, an effect which may be due to heavy metal binding in the case of the first two buffers.

DISCUSSION

The data shown in Table V, demonstrating inorganic phosphate produc- tion in excess of intermediate formation, make it evident that Reaction 1, a can only represent an over-all reaction mechanism. If phosphorylation proceeds at a rate which is actually independent of condensation, it is to be anticipated that Enzyme A may contain two components, one of which catalyzes the phosphorylation and, as the results suggest, might be present in excess. Preliminary experiments now in progress appear to support this view.

EXPERIMENTAL

Separation of Condensing Enzyme and Partial PuriJication-In the course of fractionation, the hydrolyzing enzyme is precipitated at 30 per cent ammonium sulfate saturation. The purification and properties of this en- zyme will be described in a later publication. A middle fraction, obtained between 30 and 40 per cent saturation, contains a mixture of both enzymes. At the protein concentration employed below, the bulk of the condensing enzyme precipitates between 40 and 50 per cent saturation and is entirely free of hydrolyzing enzyme activity. Table VI gives typical results of two successive fractionations. In the first, specific activity was increased from 0.8 to 5.0, with a yield of 90 per cent. A second fractionation at pH

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S. RATNER AND B. PETRACK 703

8.4 raised the specific activity further to 8.0 in Fractions 3b and 3c, with good recovery.

100 gm. of ox liver acetone powder (1) were extracted within a week of preparation with 10 volumes of 0.02 M potassium phosphate buffer, pH 7.5, at 0” with mechanical stirring for 45 minutes, and were then centrifuged at 12,000 r.p.m. for 15 minutes. The clear supernatant was brought up to 900 ml. with the same buffer. It then contained 28 mg. of protein per ml. Fractionation with ammonium sulfate was then carried out at 0” by the successive addition of 190, 63, and 63 gm., corresponding to 30, 40, and 50 per cent saturation. In each step the salt was added over a half hour inter- val and mechanical stirring was continued for a total of 2 hours before centrifuging at 12,000 r.p.m. for 15 minutes at 3”. The precipitate from

TABLE VI Purijication of Condensing Enzyme by Ammonium Sulfate Fractionation

Conditions as described in the text.

Fraction No.

Acetone powder extract

3 3a 3b 30

Salt saturation

per cent

40-50 100 O-36 32.5

3640 42.5 40-44 45.5

Volume

ml.

1800

Protein

mg. per ml.

28.0

109.0 10,900 5.0 54,500 22.8 741 5.6 4,150 54.3 2,208 8.1 17,885 68.4 2,848 8.0 23,704

Total protein Specific activity

I

w. 50,400 1.2

Total units

60,480

each cut was taken up in a minimum of 0.05 M phosphate buffer (68 ml. in all) and stored at -20” in small divided samples.

In the second fractionation, the condensing enzyme (Fraction 3) was diluted to 1 liter containing 32 ml. of molar TAM buffer, pH 8.4, and 20 ml. of 0.066 M MgS04. Fractionation was carried out as described above with successive additions of 99, 28.2, and 28.2 gm. of ammonium sulfate, corresponding to 36,40, and 44 per cent saturation. Since this was carried out without dialysis, the salt concentrations given represent the amounts added rather than those actually present. Each fraction was taken up in 30 ml. of 0.1 M TAM buffer, pH 8.4, and stored as described above.

The activity of either dilute or concentrated enzyme solutions decreases after 2 or 3 hours at O”, but is maintained without loss for at least 6 months when stored at -20” as a concentrated solution in the presence of ammo- nium sulfate. Dilutions of the enzyme were made with 0.006 M MgS04 immediately prior to testing. Since dialysis results in considerable loss of activity, most of the studies were carried out on undialyzed samples.

Methods-Enzyme activity was estimated in a medium containing the

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704 BIOSYNTHESIS OF UREA. III

following constituents per 4 ml.: 30 PM of L-citrulline, 30 pM of L-aspartic acid, 50 pM of n-3-phosphoglyceric acid, 5 PM of ATP, 26 FM of MgSO+ 200 PM of TAM buffer, pH 7.5, and 2 to 4 mg. of lyophilized muscle extract. The reaction was carried out in small test-tubes as previously described and then stopped by the addition of 8 per cent trichloroacetic acid to a final volume of 10 ml. Citrulline was estimated in 0.1 ml. of the filtrate by the method of Archibald (3). Inorganic phosphate was estimated in 0.2 ml. samples by the method of Lohmann and Jendrassik (14) and pyru- vate was estimated in 0.5 ml. aliquots by the Green, Leloir, and Nocito modification (15) of the Straub procedure (16).

Malic acid was estimated manometrically with lyophilized suspensions of malate-adapted Lactobacihs arabinosus (17, 18). Protein was estimated by a recent modification of the biuret procedure described by Gornall et al. (19). Pyrophosphate and pyrophosphatase were estimated according to Bailey (20) and myokinase activity according to Kalckar (11).

Materials-The arginase, lyophilized muscle extract, n-S-phosphogly- ceric acid, and L-citrulline were prepared as previously described. The muscle fraction employed for the ratio studies (Table V) was improved in activity by a mild heat treatment and refractionation with ammonium sul- fate before dialysis and final lyophilization. Contamination by ATPase was reduced by this procedure. L-Aspartic acid was obtained from the Nutritional Biochemicals Corporation and crude TAM, obtained from the Commercial Solvents Corporation, was recrystallized three times from al- cohol. It was adjusted to the required pH with HCI (21).

Of the various commercial ATP preparations tested, the dibarium salt purchased from the Ernst Bischoff Company gave the highest enzymatic activity. Treatment with Amberlite IR-100 was carried out as described by Polis and Meyerhof (7). In order to obtain concentrated solutions, the eluate from the column was neutralized with dilute KOH and precipitated in the cold with 3 volumes of acetone. On drying at 25” in vucuo over PzOs, the residue was dissolved in sufficient water to make a 0.05 M solu- tion, which gave the following analytical data: of the total phosphate, 3.2 per cent was inorganic, 64.5 per cent was split in 10 minutes at 100’ in 1 N

H&04, and 5.2 per cent was inorganic pyrophosphate as estimated with a specific pyrophosphatase (20). The ratio of adenine (estimated spectro- photometrically) to acid-labile phosphate was 1 .OO : 2.02.

ADP and AMP were obtained from the Sigma Chemical Company, the former as the barium salt and the latter as the crystalline free acid. The ADP was brought into solution with the aid of dilute HCl and was put on the column as a concentrated solution. The eluate and small washings were combined, neutralized, and centrifuged to remove some insoluble ma- terial, and were then about 0.03 M. 4.2 per cent of the total P was present as inorganic phosphate. The ratio of adenine to acid-labile phosphate was

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S. RATNER AND B. PETRACK 705

1 .OO : 1 .Ol . Enzymatic analysis with adenylic deaminase and myokinase (12) revealed only traces of AMP.

The AMP was dissolved with dilute KOH and was put on the column as a concentrated solution. The eluate and washings were combined. The concentration was estimated spectrophotometrically at 260 rnp and en- zymatically by means of adenylic deaminase with good agreement.

SUMMARY

1. Two enzymes which together catalyze the synthesis of arginine from citrulline by mammalian liver have been separated and partially purified.

2. A method has been developed for following the activity of the enzyme which catalyzes the condensation of citrulline with aspartic acid in the presence of ATP and Mg++.to form an intermediate product.

3. The transfer of phosphate from ATP appears to be more rapid than condensation.

4. The hydrolytic cleavage of the isolated intermediate to arginine and malic acid has been shown to be catalyzed by the second enzyme and the nature and the homogeneity of the intermediate have been demonstrated.

5. Some properties of the condensing enzyme have been described.

We are indebted to Mr. Morton C. Schneider for valuable technical assistance.

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S. Ratner and Barbara PetrackSYNTHESIS FROM CITRULLINE

FURTHER STUDIES ON ARGININE BIOSYNTHESIS OF UREA: III.

1951, 191:693-705.J. Biol. Chem. 

  http://www.jbc.org/content/191/2/693.citation

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