[34] Argininosuccinate synthetase (steer liver)

29 8

[34]

ARGININE AND ORNITHINE

[34] Argininosuccinate

Synthetase

By SARAH NH2

COOH

I

II

H2NCH

I NH

CH2

I

I + ATP

I

(CH2)3

COOH

I

t

NH

I (CH2)a

I CH2

+ AMP + PP,

I COOH

I

H2NCHCOOH L-Citrulline

COOH

C--NH----CH

I +

RATNER NH

I

1 (Steer Liver)

HzNCHCOOH L-Aspartic acid

L-Argininosuccinic acid

Assay M e t h o d la'u

Principle. The formation of AMP in the forward reaction is coupled to the oxidation of DPNH through lactic dehydrogenase and an ATPregenerating system. Pyruvate kinase, myokinase, and lactic dehydrogenase are used in excess in the presence of ATP and phosphoenolpyruvate. Inorganic pyrophosphatase is also added in excess since PPi strongly inhibits the reaction in the forward direction. Reagents and Compositionof Pool a. b. c. d. e. f. g.

L-Citrulline, 0.1 M; 0.50 ml Tris-HCl buffer, 0.1 M, pH 7.5; 1.0 ml Disodium ATP, 0.05 M; 0.20 ml MgCI2, 0.10M; 0.50 ml KCI, 1.0M; 0.20 ml Potassium phosphoenolpyruvate, 0.05 M; 0.30 ml Crystalline suspension of pyruvate kinase, 10 mg/ml, diluted 10-fold; 0.30 ml h. Crystalline suspension of lactic dehydrogenase, 5 mg/ml, diluted 10-fold; 0.20 ml The pool is made up to a final volume of 8.0 ml with 4.8 ml of water. i. Inorganic pyrophosphatase, diluted 10-fold IEC 6.3.4.5; L-Citrulline : e-aspartate ligase (AMP). taA. Schuegraf, R. Warner, and S. Ramer, J. Biol. Chem. 235, 3597 (1960). 20. Rochovansky and S. Ramer,J. Biol. Chem. 242, 3839 (1967).

[34]

ARGININOSUCCINATE SYNTHETASE j. k. /. m.

299

Crystalline suspension of myokinase, 10 mg/ml, diluted 10-fold Disodium DPNH, 0.4% L-Asparate, 0.10 M, neutralized with KOH solution Potassium phosphate, 0.02 M, pH 7.5, for dilution of all enzymes used for assay

Preparation of Reagents. Phosphoenolpyruvate is synthesized as the cyclohexylamine salt by the method of Clark and Kirby 3 and converted to the potassium salt by passage through a column of Dowex 50-K +. Inorganic pyrophosphatase (specific activity 57,000) from dried bakers' yeast was purified through the alcohol step as described/ After precipitation with (NH4)2SO4 (0-65% saturation), it was stored at - 2 0 ° at a concentration of 23.4 mg of protein per milliliter. The other three enzymes were fresh crystalline suspensions obtained from Boehringer. Procedure. The rate of DPNH oxidation is followed from the decrease in absorption at 340 mtz in a Beckman or Zeiss spectrophotometer with ultraviolet optics. To each of two cuvettes of 1-cm light path, add the following; 0.8 ml of the pool (reagents a through h), 0.02 ml o f / a n d of j, 0.5-1.0 unit of suitably diluted argininosuccinate synthetase (usually in 0.05 ml), and water to make a final volume of 1.0 ml, after all additions. The reaction cuvette, only, then receives 0.03 ml of reagent k and the optical density is noted after the reading remains unchanged for 1 or 2 minutes. The reaction is initiated by the addition of 0.05 ml of 0.I M L-aspartate (to both cuvettes). Five or six readings are taken at halfminute intervals against the "blank" cuvette which contains all the constituents except DPNH, and then the temperature of the mixture is taken. The rate remains linear for about 5 minutes. In kinetic measurements conducted for mechanism studies or other special purposes, only extensively purified enzyme should be used and the reaction should be initiated with enzyme, rather than with one of the substrates/ Definition of Unit and Calculation of Activity. One unit of enzyme catalyzes the formation of 1 micromole of product per hour at 38 °. Specific activity is expressed as units per milligram of protein. The specific activity is calculated from the expression: A OD x 60 x Ts 2 x 6.3 x mg protein where A OD represents the decrease per minute in absorbance at 3V. M. Clark and A.J. Kirby, Biochim. Biophys. Acta 78, 732 (1963). 4L. A. Heppel and R.J. Hilmoe,J. Biol. Chem. 192, 87 (1951); see also Vol. If [91].

300

ARGININE AND ORNITHINE

[34]

340 na~; Tr is the temperature correction factor, using a Oo0 of 2.0; 6.3 is the mM extinction coefficient. The factor 2 represents the ratio of DPNH oxidized per mole of AMP formed, since myokinase is provided. Protein is estimated by the biuret method 5 for steps 1 and 2, and by absorption at 280 ml z6 for the later steps. Application of Assay to Crude Fractions. The assay can be used, at the earliest, for crude enzyme obtained in step 2, for which the blank rate of DPNH oxidation is about 10% of the aspartate-dependent rate. The blank disappears after step 3. The usefulness for the assay of fractions obtained at an early purification stage applies only to extracts of acetonetreated tissue which have been subject to fractionation with (NH4)zSO 4. The initial extract (step 1) must be assayed colorimetrically from the amount of citrulline utilized, r The conditions used by Schimke s for liver homogenates may possibly also be applicable. This measures the rate of urea formation and requires supplementation with argininosuccinase.

Purification Procedure The procedure is given here for 300 g of acetone-dried steer liver. This powder was prepared from 1500 g of fresh tissue as described. 9 The purification steps given below are based on a procedure already described 1° and includes a few recent modifications) particularly of step 3. The pH of the buffers is measured at 0.1 M and 25 °. They are made up with deionized water. The phosphate buffers are made up as the potassium salts. All manipulations are carried out at 0-4°C including centrifugation at 16,000 g. All fractions, after precipitation with (NH4hSO4, are taken up in 0.02 M potassium phosphate buffer, pH 7.5. Argininosuccinate is prepared enzymatically and isolated as the barium salt. 11 Step I. Extraction of Acetone Powder. The powder is extracted with 3 liters of 0.02 M phosphate buffer pH 7.5 by stirring for 45 minutes. After centrifugation, the supernatant fluid amounts to 2590 ml. Step 2. Fractional Precipitation with (NH4)2S04. To all the material from step 1,548 g of (NH4hSO4 is slowly added with stirring over 30 minutes. After stirring 1 hour longer, the mixture is centrifuged in the 5A. G. GornaU, C. J. Bardawill, and M. M. David,J. Biol. Chem. 177, 751 (1949); see also Vol. III [73]. 60. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). 7Vol. II, p. 359. 8R. T. Schimke,J. Biol. Chem. 237,459 (1962); see also this volume [38]. 9Vol. II, p. 358. 1°O. Rochovansky and S. Ramer,J. Biol. Chem. 236, 2254 (1961). nVol. III, p. 643.

[34]

ARGININOSUCCINATE SYNTHETASE

$01

same way. A second fraction was obtained by adding 183 g of (N H4)2SO4 to the supernatant fluid. After centrifugation, a third fraction is precipitated as before with 183 g of the salt. This fraction is taken up in phosphate buffer to give 115 ml of enzyme solution to which are added 5.0 ml of 0.2 M argininosuccinate (as the potassium salt) and 5 ml of 1 M K2HPOa which brings the pH to 7.0. Step 3. Negative Adsorption on DEAE-Cellulose. The ion exchanger (Bio-Rad) is washed with water and the fine particles are decanted. A portion (60 g, moist weight) is equilibrated with 600 ml o f 0.2 M KH2PO4 and then washed six times with 4-liter portions of 5 mM potassium phosphate buffer, pH 7.9 and filtered off by suction. About 2500 mg of protein (16 ml) from step 2 is dialyzed against several changes of 5 mM potassium phosphate buffer, pH 7.9, with an increase in volume.to 28 ml. About 2.5 ml (225 mg) diluted with 65 ml of 5 mM phosphate buffer, pH 7.9, is then added to 9.2 g of the moist ion-exchange agent. After 20 minutes at 4 ° with occasional stirring, the mixture is filtered by gravity through a coarse, fritted-glass funnel, and the last few drops are expressed with positive pressure. Ten such batches are prepared at a time. The pooled filtrate (about 700 ml) is treated with 25 ml of 1 M potassium phosphate buffer, pH 7.9, and 384 g of ammonium sulfate. After centrifugation the precipitate is dissolved in 6 or 7 ml of 0.05 M phosphate buffer, pH 7.9, to which 0.5 ml of 0.2 M argininosuccinate is added. This entire procedure is carried out 8 times. Step 4. Bentonite. Approximately 1600 mg of enzyme in 20 ml is dialyzed against several changes of 5 mM phosphate buffer, pH 7.5, for 3 hours; 4 ml of 0.2 M argininosuccinate, pH 7.5, is then added. The protein concentration is adjusted to 22 mg/ml with 5 mM phosphate buffer, pH 7.5, bringing the volume to 74 ml. Bentonite (3.34 g) is added, and, after 10 minutes of intermittent stirring, the mixture is centrifuged for 5 minutes. Step 5. Heat Treatment. Immediately thereafter, the supernatant solution from step 4 (66 ml), containing approximately 9 mg of protein per milliliter, is treated with 6.6 ml of a 0.02 M solution of argininosuccinic acid at pH 7.5. The 72-ml batch is held in a 61 ° water bath for 7 minutes with rapid stirring, and then quickly chilled in ice. Denatured protein is removed by centrifugation and the protein remaining in the 68 ml of supernatant fluid is precipitated by the addition of 37 g of ammonium sulfate. The precipitate is dissolved in 5 or 6 ml of 0.1 M Tris buffer, pH 7.8. Steps 4 and 5 were carried out, on this scale, four times. Step 6. DEAE-Cellulose. About 20 g of DEAE-cellulose is washed 4 times with water to remove fines. The exchanger is then suspended in several volumes of water and subjected to reduced pressure to remove CO2. The

302

[34]

A R G I N I N E AND O R N I T H I N E

material is t h e n washed 3 times with 1 liter portions o f 0.1 N N a O H , t h e n 3 times with water, a n d t h e n equilibrated with 200 ml o f 0.2 M T r i s - H C l , p H 7.9. T h i s equilibration is r e p e a t e d o n c e m o r e , the slurry is again e x p o s e d to r e d u c e d pressure, a n d finally washed 4 times each with 2 liters o f 0.02 M Tris, p H 7.9. A p p r o x i m a t e l y 35 m g ( 1-2 ml) o f the fraction f r o m step 5 are dialyzed for 4 h o u r s against 0.02 M T r i s buffer, p H 7.8, with f r e q u e n t changes a n d are t h e n a d s o r b e d o n t o a 1.0 × 22 cm c o l u m n o f DEAE-cellulose previously equilibrated as described above a n d t h e n washed with the first eluent. O n elution with 0.02 M T r i s buffer, p H 7.8, a small p e a k o f inactive p r o t e i n e m e r g e s with 15 ml. A f t e r c h a n g i n g to the second eluent, 0.04 M Tris buffer, p H 7.9, 5 ml fractions are collected. T h e e n z y m e e m e r g e s in tubes 4 a n d 5 (occasionally also tube 6). Usually 4 c o l u m n s are r u n at a time. T h e 40-ml pool is t r e a t e d with 25.6 g o f (NH4)2SO4. T h e precipitated e n z y m e is dissolved in a b o u t 4 ml o f 0.10 M T r i s buffer, p H 7.5, a n d stored in divided samples a t - 2 0 °. T h i s 4c o l u m n p r o c e d u r e is carried out 7 times. At this stage the e n z y m e is p r o b a b l y a b o u t 5 0 % p u r e a n d is entirely free o f adenylate kinase, inorganic p y r o p h o s p h a t a s e , a n d ATPase. 2'1° T h e yields a n d specific activity o b t a i n e d a f t e r each purification step are s u m m a r i z e d in the table. PURIFICATION OF ARGININOSUCCINATE SYNTHETASEa

Fractionation step

Total Specific Total Volume proteinb activityc units Recovery (ml) (mg) (units/rag) (x 10-a) (%)

1. Extraction 2590 2. Fractionation with (NH4)2SO4) 125 3. DEAE-cellulose, negative 83 adsorption 4. Bentonite 274 5. Heat treatment 30 6. Fractionation on DEAE-cellulose 7.7

86,700 19,500 6,620 2,470 1,020 323

1.8 7.0 16.9 35 60 144

156 136 112

100 87 72

86 61 47

55 39 30

aStarting with 1.5 kg of fresh tissue. bProtein figures for steps 1 and 2 are given as biuret values and those for steps 3 through 6 are based on absorption at 280 mg. The latter values are 1.12 times higher than the former. eSpecific activity is expressed as micromoles of product formed per hour per milligram of protein at 38°. The specific activity for the fractions obtained in steps 3 through 6 have not been corrected for the difference in estimation of protein.

Properties Stability during Storage? ° T h e solution o f c o n c e n t r a t e d e n z y m e obtained at the e n d o f each step can be stored in divided samples a t - 2 0 ° .

[34]

ARGININOSUCCINATE SYNTHETASE

303

Under these conditions, the enzyme, from step 2 on, retains activity for many months since argininosuccinate is present. The presence of the latter is also essential during the heat step. Concentrated enzyme obtained after step 7 may be dialyzed against 0.02 M Tris buffer, pH 7.5, and will then retain activity for several weeks if stored at 0-4 °. However, owing to the absence of (NH4)2SO4, the activity is lost on freezing. Kinetic Values.2 The Km values (M) for the substrates of the forward reaction are: 4.6 x 10 -5, L-citrulline; 3.5 X 10 -5, L-aspartate; 8.8 X 10 -4, threo-[3-methyl-L-aspartate; 3.2 x 10 -4, ATP. The values for the backward reaction are: 2.8 x 10-5, L-argininosuccinate; 2.1 x 10 -4, AMP; 7.9 X 10 -5, PPi. The compounds, o~-methyl-DL-aspartate and v-aspartate, are inhibitors of the forward reaction competitive with aspartate and have Ki values of 1.8 x 10 -3 and 2.0 x 10 -2 M respectively. Product inhibition is observed in both forward and backward reactions3 Inhibition by PPi is noncompetitive with respect to ATP, citrulline, and aspartate, the Ki values for these being 6.2 x 10-5, 7.9 x 10 -5 and 2.2 x 10-4 M. The threo and erythro forms of two aspartate analogs have been compared. TM O f these, threo-fl-hydroxy-n,L-aspartate acts as substrate and has a Km value of 4.5 × 10 -~. The erythro form acts as inhibitor, competitive with aspartate; the Ki value is 2.4 × 10 -3. The threo and erythro forms of/3-hydroxy-/3-methyl-D,L-aspartate act as inhibitors, competitive with aspartate; the K~ values are 4.5 × 10 -2 and 7.1 X 10 -4, respectively. The methyl group in the latter compound occupies the same position as the methyl group in threo-~-methylaspartate. TM Equilibrium and pH Optima. TM The reaction is strongly pH dependent and the pH optima are 8.4 and 6.2, respectively, for the forward and backward directions. The value for the equilibrium constant K' is 0.89, 8.9, and 89 at pH 7.0, 7.5, and 8.0, respectively.

t20. Rochovansky and S. Ratner, Arch. Biochem. Biophys. 127,688 (1968).