380 ENZYMES OF PROTEIN METkBOLISM [52]

[52] Glutaminase , Asparaginase , and ~ - K e t o Ac i d - co -ami dase

By ALTON MEISTER

A. L-Glutaminase from Escherichia coli

Assay Method

CONH2 COOH I I

(CH2)2 + H~O--. (CH~)~ I I

CHNH2 CHNH2 I I

COOH COOH

-}- NH3

Principle. Enzyme activity may be measured by determinations of ammonia or of L-glutamate. 1 The assay procedure described below is based on determination of the rate of ammonia liberation.

Reagent

L-Glutamine (0.08 M), freshly prepared in 0.1 M sodium acetate buffer, pH 4.9.

Procedure. Enzyme solution (0.25 ml.) and L-glutamine (0.25 ml.) are incubated for 5 to 60 minutes at 37 °. The reaction is stopped by addition of 0.5 ml. of 15% trichloroacetic acid. Ammonia is determined by aera- tion in the presence of potassium carbonate into sulfuric acid traps. ~ Blanks containing glutamine alone and enzyme preparation alone are employed.

Definition of Unit. One unit is defined as the quantity of enzyme which catalyzes the formation of 1 aM. of ammonia per hour under the assay conditions described above.

Purification Procedure ~

Step 1. Starting Material. Escherichia coli (Strain W) is grown on a medium consisting of 0.3 % KH2P04, 0.7% K~HP04, 0.05% sodium cit- rate.2 H20, 0.01% MgS04-7 H20, 0.1% (NH4)~S04, 0.2% enzymatic di- gest of casein, and 0.5% glucose. 4 After incubation at 37 ° for 18 hours,

1 Assay based on determination of L-glutamate is not reliable in crude extracts of E. coli owing to the presence of L-glutamic acid decarboxylase.

2 j . p. Greenstein and F. M. Leuthardt, J. Natl. Cancer Inst. 5, 209 (1944) ; see Vol. I I I [145] for details of NH3 procedure. Based in part on D. Rudman and A. Meister, J. Biol. Chem. 200, 591 (1953).

4 B. D. Davis and E. S. Mingioli, J. Bacteriol. 60, 17 (1950).

[52] OMEGA-AMIDASES 381

the cells are harvested in the Sharples centrifuge, washed twice with water by centrifugation, and lyophilized. The cells may be stored at - 2 0 ° for several months without loss of glutaminase activity.

Step 2. Preparation of Cell-Free Extract. The dried cells are thoroughly ground in a mortar with 3 parts of Alumina (No. A-301, Aluminum Com- pany of America) for about 30 minutes. The ground material is then vigorously shaken with 20 parts of cold distilled water on a shaking machine at 5 ° for 20 minutes, after which the mixture is centrifuged at 18,000 × g for 20 minutes. The slightly turbid cell-free supernatant con- tains 60 to 80 % of the original activity of the cells and is about twice as active as the cells on the basis of total nitrogen (Kjeldahl). 5

Step 3. Adsorption on and Elution from Calcium Phosphate Gel. A solu- tion of calcium phosphate gel (100 ml. ; dry weight, 15 mg./ml.) is cen- trifuged, and the sedimented gel is mixed with the extract obtained from 5 g. of cells. After shaking for 30 minutes at 24 to 28 °, the mixture is centrifuged at 600 × g for 20 minutes. The supernatant solution is dis- carded, and the gel is thoroughly mixed with 40 ml. of 0.1 M KH2PO4 (pit 4.5). The mixture is shaken for 20 minutes at 5 °, followed by cen- trifugation at 5 °. Two additional elutions are carried out, and the eluates are combined and lyophilized. 6 The lyophilized material may be stored at 5 ° for several months without loss of activity. Solutions of the enzyme lose considerable activity when kept at 5 ° for three weeks. The prepara- tion at this stage contains 20 to 40 % of the total activity of the extract and exhibits an activity of 6000 to 8000 units/mg, of protein nitrogen, representing a 15- to 20-fold increase in specific activity over the cell- free extract.

Step 4. Fractionation with Sodium Sulfate. The specific activity may be doubled with the loss of approximately half of the total activity by suspension of the lyophilized powder obtained in step 3 in saturated Na2SO4 solution: 50 rag. of powder is suspended in 5 ml. of Na2SO~ solu- tion, shaken for 20 minutes at 24 to 28 °, and centrifuged. The pellet, which represents the more active fraction, is dissolved iu 1 ml. of cold water. This solution loses activity after storage at 5 ° for several days.

Properties

Specificity. The following amides are hydrolyzed at rates less than 1% of that observed with L-glutamine: D-glutamine, L-isoglutamine, L-aspara- gine, n-asparagine, L-isoasparagine, L-homoglutamine (a-aminoadipamic

5 A representative fractionation is described in Table I. L-Glutamic decarboxylase may be eluted from the calcium phosphate gel by buffer of pH 5.5. 3

382 ENZYMES OF ]PROTEIN METABOLISM [52]

acid), a-ketoglutaramate, and a-ketosuccinamate. In contrast to the crude extract, the purified preparation does not decarboxylate L-glutamic acid. 6

Effect of pH. The optional pH range (in 0.1 M acetate buffer) is 4.7 to 5.1.

Other M e t h o d s of Preparation 7

The preparat ion and properties of glutaminase isolated from C. welchii 8 have been described. Aqueous extracts of certain animal tissues (e.g., liver, kidney, brain, and spleen of the rat, rabbit, and mouse) ex- hibit glutaminase act ivi ty in the presence of phosphate or arsenate2 -11

TABLE I SUMMARY OF PURIFICATION PROCEDURE FOR GLUTAMINASE

Total Total Specific volume, Units/ml., units, Nitrogen, activity, Recovery,

Fraction ml. thousands thousands mg./ml, units/mg. N %

Extract 82 1.44 118 3.66 394 - - Eluate from gel 120 0.314 37.6 0. 0485 6,470 32 Na2S04 fraction 10 1.77 17.7 0. 149 11,900 15

This enzyme (designated glutaminase I) exhibits a pH opt imum at 8.0 and has been purified three-fold from rat liver. 12 I t is associated with particulate material. A soluble preparat ion capable of deamidating glu- tamine in the presence of a-keto acids has also been obtained from rat liver. 12 This reaction appears to involve t ransaminat ion of glutamine 13 yielding a-ketoglutaramate, which is hydrolyzed by an amidase to a-ketoglutarate and ammonia. 14 A similar mechanism is involved in the a-keto acid-mediated deamidation of asparagine: x4,15 (See a-keto acid- ~-amidase, below.) A number of animal tissues possess asparaginase ac- tivity, which may be distinguished from asparagine transaminase by dif- ferential heat inactivation.

For a recent review, see C. A. Zittle, in "The Enzymes" (J. B. Sumner and K. Myrb/ick, eds.), Vol. I, Part 2, p. 922, Academic Press, New York, 1951.

8 D. E. Hughes and D. H. Williamson, Biochem. J. 51, 45 (1952). 9 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 433 (1947).

10 j. p. Greenstein and F. M. Leuthardt, Arch. Biochem. 17, 105 (1948). 11 M. Errera and J. P. Greenstein, J. Biol. Chem. 178, 495 (1949). i~ M. Errera, J. Biol. Chem. 178, 483 (1949). See also the most recent work of

M. C. Otey, S. M. Birnbaum, and J. P. Greenstein, Arch. Biochem. and Biophys. 49, 245 (1954).

is A. Meister and S. V. Tice, J. Biol. Chem. 187, 173 (1950). 14 A. Meister, J. Biol. Chem. 200, 571 (1953). l.~ A. Meister, H. A. Sober, S. V. Tice, and P. E. Fraser J. Biol. Chem. 197, 319 (1952).

[52] OMEGA-AMIDASES 383

B. L-Asparaginase from Guinea Pig Serum

Assay Method

CONH2 COOH

CH~ CH2 I + tt~O--~ I

CHNH2 CHNH~ I I

COOH COOH

+ NH3

Principle. Enzyme activity may be conveniently measured by deter- minations of ammonia.

Reagents

L-Asparagine monohydrate (0.04 M). Sodium borate buffer (0.010 M), pH 8.5.

Procedure. Enzyme solution (0.5 ml.), substrate (0.5 ml.), and buffer (1.0 ml.) are mixed and incubated for 5 to 60 minutes at 37 °. The reaction is stopped by addition of 0.5 ml. of 15% trichloroacetic acid, and am- monia is determined as described above. 2

Definition of Unit. One unit is defined as the quantity of enzyme which catalyzes the formation of 1 ~M. of ammonia per hour under the condi- tions of the assay.

Purification Procedure 16

Step 1. Fractionation with Na2S04. Fresh guinea pig serum (100 ml.) is mixed with an equal volume of 30 % Na~SQ. The mixture is allowed to stand at 24 to 28 ° for 30 minutes and then centrifuged at 1000 X g for 60 minutes. The supernatant solution is discarded, and the precipitate is dissolved in 50 ml. of cold water. This preparation is four to five times as active as the original serum.

Step 2. Adsorption of Impurities by Calcium Phosphate Gel. The final solution prepared in step 1 is mixed with the residue obtained by cen- trifuging an equal volume of calcium phosphate gel (dry weight, 15 mg./ ml.). After shaking at 24 to 28 ° for 30 minutes, the gel is removed by centrifugation. The solution may be stored in the frozen state without loss of activity over a period of several weeks.

Properties

Specificity. The purified preparation does not hydrolyze L-isoaspara- gine, L-glutamine, D-glutamine, DL-C~-methylasparagine, L-homogluta-

,6 A representative fractionation is described in Table II .

3 8 4 ENZYMES OF PROTEIN METABOLISM [52]

mine (a-aminodipamic acid), a-ketosuccinamate, and a-ketoglutaramate. I t hydrolyzes D-asparagine at about 3% of the rate observed for the L-isomer.

Effect of pH. The optional pH range (in 0.1 M sodium borate buffer) is 7.5 to 8.5.

TABLE II SUMMARY OF PURIFICATION PROCEDURE FOR ASPARAGINASE

Specific Total volume, Total Nitrogen, activity, Recovery,

Fraction ml. Units/ml. units mg./ml, units/mg. N %

Serum 100 168 16,800 8.45 19.9 - - Step 1 50 272 13,600 3.20 85.1 81 Step 2 47 247 11,600 1.72 144 69

Other Methods of Preparation 7

The preparat ion of yeast asparaginase has been described. 17

C. a-Keto Acid-~-Amidase from Rat Liver CONH2 COOH ] J I + H20-~ I

C - - O C : O I I

COOH C 0 0 H n = l , 2

Assay Method

+ NH3

Principle. The reaction is followed by determinat ion of the ammonia or a-keto acid formed.

Reagent

Sodium a-ketoglu taramate or a-ketosuccinamate (0.03 M), freshly prepared in 0.1 M sodium borate buffer, p H 9.0.

Procedure. Enzyme solution (1 ml.) and substrate (1 ml.) are incu- bated for 15 to 60 minutes at 37 ° . Ammonia is determined as described above. *

Definition of Unit. One unit is defined as the quan t i ty of enzyme which catalyzes the formation of 1 uM. of ammonia per hour under the assay conditions.

17 W. Grassmann and O. Mayr , Z. physiol. Chem. 214, 185 (1933).

[52] OMEGA-AMIDASES 385

Purif icat ion Procedure 14,1s

Step 1. Forty-f ive grams of minced fresh rat liver is blended in a Waring blendor with 90 ml. of ice-cold water for 2 minutes. After cen- tr ifugation at 600 X g at 5 ° for 2 hours, the supernatant solution is dialyzed against running water at 10 ° for 18 hours and centrifuged again.

Step 2. Differential Heat Denaturation. The solution (100 ml.) ob- tained above is heated at 60 ° 19 in a water bath for 40 minutes. The solution is cooled in ice, and the coagulated protein is removed by centrifugation.

Step 3. Adsorption on and Elution from Calcium Phosphate Gel. The solution obtained in step 2 is mixed with 67 ml. of calcium phosphate gel (15 mg. dry weight per milliliter), and the mixture is shaken for 30 min- utes at 24 to 28 °. After centrifugation, the gel is drained free of the super- na tant solution and is washed twice by centrifugation with 20 ml. of water. The gel is then shaken successively with two 20-ml. portions of 0.2 M potassium phosphate buffer (pH 5.5). The gel is t reated in the same manner successively with buffer of pH 6.0 and pH 6.5. The most active fraction is obtained by elution at pH 6.5. This solution is adiusted to pH 7.5 with 0.5 N N a 0 H , and stored at 0 ° or frozen. The preparat ion maintains its original act ivi ty for only one or two weeks. Lyophilization or dialysis results in inactivation.

TABLE III SUMMARY OF PURIFICATION PROCEDURE FOR a-KETO ACID-co-AMIDASE a

Total Specific volume, Total Nitrogen, activity, Recovery,

Fraction ml. Units/ml. units mg./ml, units/mg. N %

Liver homogenate 136 104 14,100 10.2 10.2 - - Step 1 100 87.4 8,740 4.51 19.4 62 Step 2 91 85.3 7,750 2.63 32.4 55 Step 3 (gel eluate,

pH6.5) 40 42.3 1,690 0.12 353 12

° Substrate, a-ketoglutaramate.

Propert ies 14

Specificity. The enzyme does not deamidate the optical isomers of asparagine and glutamine, L-homoglutamine, or a-ketoadipamate.

Effect of pH. The optional pH range (in 0.1 M sodium borate buffer) is 8.5 to 9.5.

is A representative fraetionation is described in Table III. ~9 A thermometer is placed in the enzyme solution and gentle stirring is carried out

throughout the heating procedure.