- Email: [email protected]
Purification and properties of d -glutamic acid oxidase from Octopus vulgaris lam
ARCHIVES
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
77, %6-%9
(1958)
and Properties of D-Ghtamic Acid Oxidase from Octopus vulgaris Lam.l E. Rocca’ and F. Ghiretti
From the Department
of Physiology,
Stazione
Zoologica,
Naples,
Italy
Received January 2, 1958
Blaschko and Hawkins (1) first found that extracts of cephalopod hepatopancreas contain an oxidase which acts on many n-amino acids. It was observed that n-glutamic acid was one of the amino acid oxidized. Later the same authors, by mixed substrate experiments, showed that the hepatopancreas of several cephalopods contains two enzymes, one general n-amino acid oxidase with a specific pattern similar to that of mammal oxidase, and a second enzyme which oxidizes n-glutamic acid (2). The latter also acts upon n-aspartic acid, but at a slower rate. The present paper deals with the purification of the n-glutamic oxidase from the octopus hepatopancreas and with the study of its chemical properties. EXPERIMENTAL Reagents n-Glutamic acid (Fluka) and n-aspartic acid (National Biochemicals Co.) were converted in sodium salts. All the other amino acids were National Biochemicals Co. products. cY-Ketoglutaric acid was twice crystallized from glacial acetic acid, benzene, and petroleum ether. Pyruvic acid (Merck) was distilled under vacuum, and the concentration of the solution used was determined by titration. Flavine adenine dinucleotide (FAD), 8070 pure, flavine mononucleotide (FMN), and riboflavine were Sigma products. The solutions were kept frozen at -20°C. Monoiodoacetic (British Drug House), o-iodosobenzoic, and p-chloromercuribenzoic acids (Sigma) were neutralized with NaOH before use. Calcium phosphate gel was prepared as described by Keilin and Hartree (3). 1 Aided by a grant (RG-4845) of the U. S. Public Health Service. 2 Present address: University of Milan, Department of Zoology and Comparative Anatomy. 336
D-GLUTAMIC
ACID
OXIDASE
337
Methods Preliminary experiments showed that hydrogen peroxide is formed in the oxidase reaction and affects the enzyme activity. Excess catalase was added to insure complete destruction of the Hz02 in all tests. Neither FAD nor FMN was found to be required for the activity of the enzyme. It was observed that the rate of oxidation in oxygen compared with air increased by 7OoJ,. The activity of the enzyme was measured by the rate of oxygen consumption in the conventional Warburg apparatus at 35°C. The vessels contained 1 ml. of 0.1 M pyrophosphate buffer pH 8.3, crystalline catalase (equivalent to about 100 units) dissolved in 0.2 M Tris buffer pH 8.3, an amount of enzyme to give 80-150 ~1. 02 uptake in 30 min., and water to a final volume of 3.0 ml. Different amounts of substrate (30 pmoles n-amino acids, 60 pmoles m-amino acids, 300 pmoles glycine) were put in the side arm. KOH was routinely present in the center cup. The assembled manometers were gassed with oxygen for 5 min. and then shaken at 35°C. After 34 min. equilibration, the reaction was started by adding the substrate from the side arm. After a further 5 min. of shaking, the first reading was made. Subsequent readings were taken at 5-min. intervals for 30 min. All the experiments were run in duplicate. As a measure of purification, an arbitrary enzyme unit was used which is defined as the amount of enzyme which gives 1 ~1. oxygen uptake in 30 min. Specific activity was calculated as units/mg. protein. The protein concentration was determined spectrophotometrically according to Kalckar (4). Catalase was crystallized from beef liver according to Mosimann (5). Its activity was determined with the method of von Euler and Josephson (6). Ammonia was estimated by nesslerization after steam distillation; cY-ketoglutaric acid and pyruvic acid were determined calorimetrically according to Friedmann and Haugen (7). Further identification of the keto acids was made by paper chromatography (8-10) of their phenylhydrazones prepared according to Cavallini and Frontali (11). Oxalacetic acid, when present, was determined as pyruvic acid after decarboxylation with nickel sulfate.
Puri$cation
of o-Glutamic
Acid Oxidase
The hepatopancreas of Octopus vulgaris were collected over a period of 2-3 months and kept frozen at -20°C. The organs were homogenized in a Waring blendor with cold acetone (- 20°C.) ; the suspension was filtered in a Biichner funnel and washed with cold acetone until the effluent liquid was colorless. From 10 to 15 vol. acetone was used per weight of organ. The acetone powder was dried at room temperature and kept at 4°C. in a desiccator over CaClz for no longer than 2 weeks (Table I). Step 1. The powder was carefully suspended in 15-20 vol. of 0.05 M pyrophosphate buffer pH 8.3 and extracted for 1 hr. with continuous stirring over a magnetic stirrer. This and all further operations were
338
ROCCA
AND
GHIRETTI
TABLE I f’uri$cation
of D-Glutamic
Acid.
Oxidase
5 K.-of acetone lwder) -(2 -
step -
Vol. of solution
Total units
420 220
82,000 28,600
-~~mg./ml. 67.5 4.25
220 34
22,400 12,800
1.25 1.37 0.04
ml.
1. Aqueousextract
2. First (NH&SO, precipitate 3. Acid precipitate 4. Second (NH1)804 precipitate 5. Ca gel treatment
-
10
-
1,230
70 2.89 30.4
10.5
35
81 274
28 95
27 15.6
3200
1100
1.6
made at 4°C. The suspensionwas centrifuged at 2000 X g for 30 min., and the solid material was discarded. The aqueous extract was found to be much more stable than the acetone powder; it can be kept frozen for 2-3 months without any lossof the activity. Specific activity was 2.9. Step 6. First precipitation with (NH&S04. For each 100 ml. of crude extract 14.4 g. of solid (NH&SOI was added very slowly and, after 30 min. of continuous stirring, the precipitate formed was centrifuged off. A further 11 g. of (NH,),S04/100 ml. was added to the clear supernatant, and the precipitate, which contains the enzyme, was collected by centrifugation, dissolved in 0.1 M pyrophosphate pH 8.3 and dialyzed for 12 hr. against 0.01 M Na2HP04 plus 10e3M ethylenediamine tetraacetate (EDTA) (Versene). Specific activity, 30.4; yield, 35%. Step S. Precipitation at pH 4.8. The liquid was brought to pH 4.8 with 2 N acetic acid and immediately centrifuged at 3000 X g for 15 min. The precipitate was discarded and the supernatant neutralized with 1 N NaOH. Specific activity, 81; yield, 27%. Step 4. Second precipitation with (NH&S04 . To the neutralized supernatant, 17.6 g. (NH&SO&O0 ml. was added slowly and the precipitate was centrifuged off. To the supernatant, a further 11 g./lOO ml. was added. The precipitate was collected by centrifugation, dissolved in 0.1 M pyrophosphate buffer pH 8.3, and dialyzed against 0.01 M NazHP04 plus EDTA for 12 hr. Specific activity, 274; yield, 15.6 %. This preparation was used for most of the experiments. Step 5. Treatment with calcium phosphate gel. The solution obtained in Step 4 was brought to pH 6.8 by dialyzing for 12 hr. against
D-GLUTAMIC
ACID
339
OXIDASE
1O-3 M phosphate buffer, then treated with calcium phosphate gel (0.0317 g./ml.). To each 20 ml. extract, 0.3 ml. Ca gel was added, and after 20 min. the sediment was centrifuged off. This treatment was repeated twice. The clear supernatant was then stirred with a further 2.0 ml. Ca gel which adsorbed entirely the enzyme. After washing with 1O-3 M phosphate buffer pH 6.8, the enzyme was eluted with 1O-2 M of the same buffer at pH 6.8. Specific activity, 3200; yield, 1.6 %. Properties
of D-Glutamic Acid Oxidase
Stoichiometry of the Reaction. In the presence of catalase, 1 mole of oxygen is required for the oxidation of 2 moles of n-amino acid. The relationship between oxygen uptake and formation of ammonia and keto acids was studied during the oxidation of n-glutamic acid and of n-aspartic acid. Warburg vessels with two side arms were used. Readings were taken immediately after addition of the substrate. After a certain period the reaction was stopped with 0.3 ml. of 60% perchloric acid. The contents of the vessels were cooled in ice for 10 min., then filtered, and ammonia and keto acids were determined in the deproteinized fluid. The data shown in Table II are in quite good agreement with the theoretical values. Further identification of the keto acids was obtained by paper chromatographic separation of the hydrazones formed (Table III). The oxidstion product of n-aspartic acid, viz., oxalacetic acid, was identified as pyruvic acid after treatment with 0.1 M (final concn.) ?XK~, TABLE II Stoichiometry of the Reaction The vessels contained: 1 ml. purified enzyme (1.0-1.5 mg. protein); cat&se 100 units; 0.3 ml. of 0.1 M pyrophosphate buffer pH 8.3; 30 rmoles substrate; and 0.1 ml. KOH in the center well. The final volume was made up to 3.1 ml. with water. Gas phase, oxygen. Temp., 35°C. Time of the experiment 60 min.
Substrate
n-Glutamic n-Aspartic
acid acida
0% uptake
NHa. productmn
______ ~moles
pmoles
12.1 11.2
23.7 24.2
Keto acid production a-Kg. ~___ pnmles
- 02 NH3
K-acid NH0
K-acid 02
1.08 1.06
2.12 2.31
Pyr. -/moles
25.6 25.9
0.51 0.46
a The oxalacetic acid formed was determined as pyruvic 0.1 M (final concn.) NiSO, for 30 min. at room temperature.
after treatment
with
340
ROCCA
AND
GHIRETTI
TABLE III Chromatographic Identijkation of the Keto Acids in the Reaction Fluid after Oxidation of D-Gktamic and n-Aspartic Acids by n-Glutamic Oxidase The reaction mixture was deproteinized at 0°C. with 6% (final concn.) HClO, and filtered; the keto acids were converted in phenylhydraeones and extracted according to Cavallini and Frontali (12). When n-aspartate was the substrate, oxalacetic acid was decarboxylated with NiSO, . Whatman paper No. 1. Temp., Paper
27T.
-
Reaction
a-Kg.
Method
Pyr. D-Glut.
_Rf
Ascending chromatography. 12 hr. butanolEtOH-NH3 (10:20:0.5 N) El Hawary (8). Descending chromatography. 4 hr. Paper washed with 0.1 M phosphate buffer pH 7.2 pet.rol. ether-EtOH (20:80) Kun (9). Descending chromatography. 13 hr. Chamber saturated with EtOH 95%. EtOH-water (83:17) Kun (9). Ascending chromatography. 4 hr. Chamber saturated with phenol. glycineNaOH 0.1 M pH 8.4 Virtanen (10).
RJ
0.03
0.47 0.69 0.17 0.40
0.50 0.39
0.13
RJ
-
mixture
-
n-Asp. RJ
0.03
0.20 0.47 0.05 0.17
0.73 0.82
0.51
0.55 0.70
0.44
0.39
0.45
-
0.13
-
TABLE IV of Amino Acids by Crude Extracts of Octopus Hepatopancreas The vessels contained: 1 ml. crude extract; 0.3 ml. 0.1 M pyrophosphate buffer pH 8.3; 0.3 ml. substrate (30 pmoles n-amino acids, 60 pmoles m-amino acids, 300 pmoles glycine); 0.1 ml. 15% KOH in the center well. The final volume was made up to 3.1 ml. with water. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 60 min. Oxidation
Relative
Substrate
n-Glutamate n-Aspartate Glycine n-Isoleucine nn-Isoleucine nn-Alanine n-Asparagine nn-or-Aminobutyric n-Glutamate L-Aspartate
acid
activity
100 65 43 31 31 67 74 171 0 0
D-GLUTAMIC
ACID
341
OXIDASE
Enzyme Specificity. The crude extract (Step 1) was found active on a number of o-amino acids and on glycine. L-Amino acids were not oxidized (Table IV). The activity on glycine was lost after the first (NH&SO, precipitation (Step 2). After treatment at pH 4.8, only the activity on u-glutamate and to a lesser degree on u-aspartate was retained. Neither FAD nor FMN addition was able to restore the activity on the other amino acids. This demonstrates that D-glutamic acid oxidase is not identical with the mammal D-amino acid oxidase. The ratio of activity on n-aspartate to activity on D-glutamate was also investigated. It remained constant during the purification procedure. This is an indication that the oxidation df the two amino acids is catalyzed by the same enzyme (Table V). Effect of Enzyme Concentration. The activity of the enzyme toward I,-glutamic acid was found to be proportional to the amount of protein used, when the substrate concentration was maintained constant (Fig. 1). Eflect of Substrate Concentration. The oxygen uptake was determined for various concentrations of n-glutamic and u-aspnrtic acids at pH 8.3 and 35°C. The results, plotted by the method of Lineweaver and Rurk, are represented in Fig. 2. The Michaelis constant is 8 X 1OP Al and 4.5 X lo-” ill for r)-glutamic acid and u-aspartic acid, respectively. TABLE
of
Activity
D-Glutamic
V
Acid Oxidase on n-Aspartic Acid Various Degrees of Purification
and
D-Glutarnic
Acid
at
The reaction mixture contained: 0.5 ml. extract; 0.3 ml. of 0.1 M pyrophosphate buffer pH 8.3; 0.3 ml. substrate (30 pmoles), urater to a final volume of 3.0 ml.; 0.1 ml. KOH 15yo in the center well. The oxygen uptake was followed for 30 min. Gas phase, oxygen. Temp., 35°C. ___Experiment
Iio.
step
D-Aspartic
@l. 02/30
I
II
ac.
D-Glutamic
ac.
B
A ?nin
pl. 01j30
A F
min
1 2 3 4
77.7 121 .o 187.0 239.0
120.0 159.6 152.4 304.0
0.65 0.75 0.70 0.78
1 2 3 4
31.4 53.2 30.2 66.0
61.0 80.0 23.6 100.0
0.51 0.67 0.68 0.66
342
ROCCA
AND
GHIRETTI
E$ect of pH. pH and buffer markedly affected the rates of oxidation of n-glutamic and n-aspartic acids. In Table VI the rates obtained in different buffers are reported. The strong inhibition by Verona1 was further investigated and compared with chemically related compounds (see below). The pH optimum was investigated using pyrophosphate buffer. In acid solution the activity of the enzyme decreases rapidly. n-Glutamic acid and n-aspartic acid have similar pH curves with the same maximum: 8.1-8.3 (Fig. 3). Coupled Oxa’dation with Ethyl Alcohol. Keilin and Hartree (12) showed that an oxidase system which forms hydrogen peroxide can be coupled with the oxidation of other substances such as ethyl alcohol. When the oxidation of an amino acid is carried out in the presence of EtOH, the rate of oxygen uptake is doubled. The oxidation of n-glutamic acid and
I
I
20 -
FIG. 1. Effect of enzyme concentration on the oxidation of n-glutrtmic acid. The Warburg vessel contained: different amounts of purified enzyme (Step 4); 1 ml. of 0.1 M pyrophosphate buffer, pH 8.3; catalase 100 units; 30 amoles Dglutamic acid; and water to a final volume of 3.0 ml.; 0.1 ml. KOH 15% in the center well. Gas phase, oxygen. Temp., 35°C. Ordinate, pl. oxygen uptake in 30 min. Abscissa, mg. protein.
D-GLUTAMIC I
,
,
,
,
I
,
1
ACID
343
OXIDASE
,
70 50 5c 4c JO 20 10 1
I
I
I
,
I
I
I
I
I
500
1
I
I
I
I
I
1
250
th FIG. 2a. FIG. 2. Velocity of oxidation of n-glutamic acid n-glutamic acid oxidase at pH 8.3 and temp. 35%. l/v of oxygen uptake expressed in ~1. 02 for 15 min.; l/S centration of n-glutamic acid expressed in moles/l. aspartic acid.
[sj FIG.
I
I
50‘0 .
2b.
and of n-aspartic acid by is the reciprocal of the rate is the reciprocal of the con(a) n-glutamic acid. (b) D-
TABLE VI on D-Ghtamic Acid Oxidase The incubation mixture contained: 0.3 ml. enzyme extract (Step 4) = 0.27 mg. protein; 0.3 ml. n-glutamate (30 pmoles) ; catalase 100 units; 1 ml. of 0.1 M buffer pH 8.3; and water to a final volume of 3.0 ml. Gas phase, oxygen. Temp., 35°C. Effect
.-I_
of Buffers
BUffW
Pyrophosphate Trisa Borate Ammediolb Verona1 a Tris(hydroxymethyl)aminomethane. * 2-Amino-2-methyl-l, 3-propanediol.
~1. O&Z0 min.
87.0 105.0 190.5 97.0 12.9
Relative
activity 100
121 115 112 15
344
ROCCA
AND
GHIRETTI
of nn-cr-aminobutyric acid by crude extract of octopus hepatopancreas was studied with and without a definite amount of EtOH. With eit,her substrate, the rate of oxygen uptake was doubled in the presence of EtOH. Inhibition by Benxoate. Klein and Kamin (13) first noticed that sodium benzoate in 1O-3 M concentration completely inhibits the Damino acid oxidase from mammals. We found that, whereas benzoate inhibited by 33 % the residual n-amino acid oxidase present in the crude extract of octopus hepa~pancreas, it had no effect on the purified n-glutamic enzyme. Inhibitian by Verona1 and Related Compounds. Verona1 was found to have a strong inhibitory effect on the oxidase system. Of the other relate compounds tested, only urethan inhibited (Table VII). Urea and barbituric acid were not effective. It may be remembered that Verona1 buffers are known to inhibit the activity of glycine oxidase almost completely (14).
I 80-
I
I / “\
%
$40 -T *+Oi / 20- o 0 ’ 6.0 FIG.
30,.
8 x0 FIG.
I 8.0
I.
I
8 9.0
t a0
3b.
3. Oxidation of n-glutamic acid and of n-aspartic acid by n-glutamic acid oxidase as a function of pH. All the experiments were made with 0.3 ml. purified enzyme (Step 4); 1.5 ml. of 0.1 M pyrophosphate buffer; 100 units of catdase; 30 pmoles substrate; and water to a final volume of 3.0 ml.; 0.1 ml. KOH 15% in the center well. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 30 min. The pH’s were controlled at the end of the experiment. (a) n-glutamic acid. (5) n-aspartic acid. FIG.
ph
D-GLUTAMIC
ACID
TABLE Effect
of
Sodium
345
OXIDASE
VII
Diethylmalonylurea (Veronal), Malonylurea on the D-Glutamic
Urea, Urethan Acid Oxidase
and
Sodium
The reaction mixture contained: purified enzyme Step 4 (0.3 mg. protein); 0.03 M (final concn.) pyrophosphate buffer pH 8.3; 30 pmoles n-glutamic acid; catalase 100 units. Total volume 3.0 ml.; KOH 15yo 0.1 ml. in the center well. Gas phase, oxygen. Temp., 35°C. Inhibitor
02 uptake
Concentration M
None Verona1 Verona1 Verona1 None Urea Urethane Na malonylurea
p1./30 min.
1.6 X 1O-3 8.0 X 1O-3 1.6 X 1OP 1.0 1.0 1.0
x x x
10-Z lo-2 10-z
91.7 74.2 36.6 18.8 73.7 78.7 50.8 88.0
Inhibition %
19 60 75 0 31 0
Inhibition by Suljhydryl Reagents. The importance of -SH groups for the full activity of mammal n-amino acid oxidase has been well established a long time (15, 16). It was also found that complete reversal of inhibition was produced by addition of glutathione. Similarly, n-glutamic acid oxidase was strongly inhibited by monoiodoacetat’e, o-iodosobenzoate, and p-chloromercuribenzoate. The latter showed the strongest inhibitory action: 74% inhibition with 1O-5 M. Twenty equivalents of cysteine produced complete reversal of inhibition (Table VIII). Inhibition by L-Glutamic Acid. As previously stated, L-amino acids are not oxidized either by the crude extract or by the purified enzyme. Moreover, it was found that L-glutamic acid inhibits the oxidation of n-glutamic acid. The nature of this inhibition was investigated using varying amounts of substrate and of inhibitor. The results, shown in Fig. 4, indicate the competitive nature of the inhibition. Characterization of the Prosthetic Group. The spectrophotometric study of the purified enzyme did not give satisfactory results. Attempts to resolve the enzyme into apoenzyme and prosthetic group by the method described by Warburg and Christian (17) failed. Better results were obtained with prolonged dialysis against acid (KH&SO~ . The buffer used was citrate-phosphate containing 24.3 % (NH&S04 . The pH value of the solution appeared to be critical. Treatment for 2-3 hr. at pH 3.8 gave only partial inactivation of the enzyme. In all cases the
346
ROCCA
AND
GHIRETTI
TABLE VIII of -SH Reagents and Reactivation with Cysteine The reaction mixture contained: purified enzyme 0.58 mg. protein (Step 4); 0.03 M (final concn.) pyrophosphate buffer pH 8.3; catalase 100 units; 30 rmoles n-glutamic acid. Total volume 3.0 ml. with water; KOH 15yo 0.1 ml. in the center well. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 30 min. Inhibition
Inhibitor
Inhibition %
Monoiodoacetate
lXWBM 1 X 10-b M 1X lO-‘M 1 X 10-4 M plus cysteine 2 X 10-z M
p-Chloromercuribenzoate
1 1 1 1
o-Iodosobenzoate
0 48 68 18
W6 M lo-6 M lO-‘M W4 M plus cysteine 2 X W2 M
27 74 100
lXlO+M 1 X 1P M 1X10-‘M 1 x 10-a M 1 X lOma M plus cysteine 2 X 1P M
8 21 32 52
X X x X
0
0
was restored by addition of FAD. Longer treatment at, the same pH gave greater but irreversible inactivation. Treatment of the enzyme at, lower pH (2.8) for 3 hr. produced in most casescomplete loss of activity (Table IX). Neither FMN nor riboflavine were found to be able to replace FAD for the reactivation of n-glutamic acid oxidase. activity
COMMENTS
The results of the experiments described in this paper demonstrate that the enzyme purified from the hepatopancreas of Octopus vulgaris is a specific oxidase for n-glutamic acid. The identity of n-glutamic enzyme with the mammalian n-a,mino acid oxidase can be excluded since this enzyme does not attack n-glutamic acid and since the two oxidases that are present in octopus hepatopancreas can be easily separated during the purification procedure. In addition to this, sodium benzoate, which is known to be a specific inhibitor for the mammal n-amino acid oxidase, has no effect, on the n-glutamic enzyme.
D-GLUTAMIC
ACID
OXIDASE
FIN. 4. Competitive inhibition of n-glutamic acid oxidation acid. l/v represents the reciprocal of the rate of oxygen uptake oxygen/l5 min.; l/S represents the reciprocal of the concentration acid expressed in molarity. 1. No L-glutamate present. K, = 6 X 10e3M. 2. 0.1 M n-glutamate. K, = 11 X 1OP M. 3. 0.2 M L-glutamate. K, = 19.5 X lo+ M. The system contained also: 0.03 M (final concn.) pyrophosphate cntalase 100 units; different amounts of n-glutamic acid; and volume of 3.0 ml. Gas phase, oxygen; temp., 35°C.
347
by L-glutamic expressed in ml. of o-glutamic
buffer pH 8.3; water to a final
n-Glutamic acid oxidase is also active, but at a slower rate, on n-aspartic acid. That the oxidation of the two dicarboxy-u-amino acids is catalyzed by the same enzyme is demonstrated by the oxidation ratio for the two substrates, which remains constant during the purification. Green et al. (18) prepared from the “cyclophorase” system of mammals an enzyme specific for u-aspartic acid. This enzyme has in common
348
ROCCA
AND
GHIRETTI
TABLE Reactivation
with
FAD
IX
of u-Glutantic
Acid
Oxidase
after
Acid
l’reatment
The purified enzyme (Step 4) was dialyzed in the cold (4°C.) against 50 vol. of 0.05 M citrate and NazHPOI buffers containing 24.3% (NH&SO* . The precipitate was collected by centrifu~ation at 4000 X g for 5 min., washed, and redissolved in 0.1 M pyrophosphate buffer pH 8.3. The reaction mixture contained: enzyme 0.5 ml.; 1.0 ml. of 0.1 M pyrophosphate buffer pH 8.3; catalase 100 units; 0.3 ml. of 10-d M FAD; 30 pmoles n-glutamio acid. Final volume 3.0 ml. with water, 0.1 ml. KOH 15% in the center well. The readings were taken after 20 min. of incubation. Gas phase, oxygen. Temp., 35°C. Acid Enzyme
treatment
pl. Oa/30 min.
preparation
IV-2 V-l V-l VI-2 VIII-1
PH
hr.
No FAD
3.8 3.8 3.8 2.8 3.8
2 3 8 2 2
40.6 45.0 9.3 5.4 66.5
With
FAD -
76.0 80.5 36.8 26.0 136.7
with the octopus n-glutamic enzyme the insensitivity to benzoic acid, but differs from it with respect to its substrate specificity. As for the inhibitory action of Verona1 and urethan, it must be remembered that Verona1 inhibits also the activity of glycine oxidase almost complexly (14). The experiments of reactivation after acid treatment indicate that n-glutamic acid oxidase from octopus is a flavoprotein. Flavoproteins are known to produce hydrogen peroxide when reacting with molecular oxygen. It follows therefore that the ratio of oxygen uptake in the absence and in the presence of catalase is 2: 1. The relatio~~p between oxygen uptake and substrate oxidized when excess of catalase was present, demonstrated that hydrogen peroxide is produced during the oxidase reaction. The formation of peroxide was further demonstrated by the possibi~ty of coupling the oxidative deamination of n-glutamate or n-aspartate with the oxidation of ethyl alcohol. Under the conditions used in this study, n-glutamic acid oxidase could be partly resolved into apoenzyme and prosthetic group; reactivation occurred when the dialyzed preparation was supplemented with FAD. Other prosthetic groups like F&IN or riboflavine cannot replace FAD. The difficulty with which the n-glutamic enzyme is split under acid conditions, compared with the relative mild conditions employed for the mammal n-amino acid oxidase and the n-aspartic
D-GLUTAMIC
340
ACID OXIDASE
acid oxidase, can be regarded as evidence that in octopus enzyme the prosthetic group is more firmly bound to the npoenzyme.
The authors script.
wish
to thank
Prof.
8. M.
Rapoport
who
kindly
revised
the manu-
SUMMARY 1. A procedure is described for the purification of wglutamic acid oxidase from the hepatopancrens of Octopus vulgaris. 2. Optimum conditions are given for the assay of the enzyme. The activity is higher in oxygen than in air. The influence of buffers, of enzyme concentration, and of substrate concentration is studied. The ratio of oxidation for n-glutamic and n-aspartic acids is presented together with the relative rates of oxidation of other amino acids. 3. The nature of the prosthetic group has been investigated. The experiments of reactivation after acid treatment indicate that the enzyme is a flavoprotein with flavine adenine dinucleotide as the prosthetic group.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. !). 10. 11. 12. 13. 14. 15. 16. 17. 18.
BLASCHKO, H., AND HAWKINS, J., Biochem. J. 62, 306 (1952). BLASCHKO, H., AND HIMMS, J. M., J. Physiol. (London) 128, 7P (1955). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) Bl24, 397 (1938). KALCKAR, H. N., J. Biol. Chem. 167, 461 (1947). MOSIMANN, W., Arch. Biochem. Biophys. 33, 487 (1951). EULER, H. VON, AND JOSEPHSON, K., Ann. 462, 158 (1927). FRIEDMANN, E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). EL HAWARY, F. S., AND THOMPSON, R. H. S., Biochem. J. 63, 34 (1953). KUN, E., AND GARCIA-HERNANDEZ, M., Biochim. et Biophys. Acta 23, 181 (1957). VIRTANEN, A. I., MIETTININEN, J. K., AND KUNTNER, H., Beta Chem. Scar&. 7, 38 (1953). CAVALLIXI, D., AND FRONTALI, M., Biochim. et Biophys. Acta 13, 459 (1953). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) B119, 114 (1936). KLEIN, J. It., AND KAMIN, H., J. Biol. Chem. 138, 507 (1941). RATNER, S., NOCITO, V., AND GREEN, D. E., J. Biol. Chem. 162, 119 (1944). SINGER, T., AND BARRON, E. S. G., J. Biol. Chem. 167, 241 (1945). SINGER, T., J. Biol. Chem. 174, 11 (1948). WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 298, 150 (1938). STILL, S., BOELL, M. V., KNOX, W. E., AND GREEN, D. E., J. Biol. Chem. 179, 831 (1949).
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
77, %6-%9
(1958)
and Properties of D-Ghtamic Acid Oxidase from Octopus vulgaris Lam.l E. Rocca’ and F. Ghiretti
From the Department
of Physiology,
Stazione
Zoologica,
Naples,
Italy
Received January 2, 1958
Blaschko and Hawkins (1) first found that extracts of cephalopod hepatopancreas contain an oxidase which acts on many n-amino acids. It was observed that n-glutamic acid was one of the amino acid oxidized. Later the same authors, by mixed substrate experiments, showed that the hepatopancreas of several cephalopods contains two enzymes, one general n-amino acid oxidase with a specific pattern similar to that of mammal oxidase, and a second enzyme which oxidizes n-glutamic acid (2). The latter also acts upon n-aspartic acid, but at a slower rate. The present paper deals with the purification of the n-glutamic oxidase from the octopus hepatopancreas and with the study of its chemical properties. EXPERIMENTAL Reagents n-Glutamic acid (Fluka) and n-aspartic acid (National Biochemicals Co.) were converted in sodium salts. All the other amino acids were National Biochemicals Co. products. cY-Ketoglutaric acid was twice crystallized from glacial acetic acid, benzene, and petroleum ether. Pyruvic acid (Merck) was distilled under vacuum, and the concentration of the solution used was determined by titration. Flavine adenine dinucleotide (FAD), 8070 pure, flavine mononucleotide (FMN), and riboflavine were Sigma products. The solutions were kept frozen at -20°C. Monoiodoacetic (British Drug House), o-iodosobenzoic, and p-chloromercuribenzoic acids (Sigma) were neutralized with NaOH before use. Calcium phosphate gel was prepared as described by Keilin and Hartree (3). 1 Aided by a grant (RG-4845) of the U. S. Public Health Service. 2 Present address: University of Milan, Department of Zoology and Comparative Anatomy. 336
D-GLUTAMIC
ACID
OXIDASE
337
Methods Preliminary experiments showed that hydrogen peroxide is formed in the oxidase reaction and affects the enzyme activity. Excess catalase was added to insure complete destruction of the Hz02 in all tests. Neither FAD nor FMN was found to be required for the activity of the enzyme. It was observed that the rate of oxidation in oxygen compared with air increased by 7OoJ,. The activity of the enzyme was measured by the rate of oxygen consumption in the conventional Warburg apparatus at 35°C. The vessels contained 1 ml. of 0.1 M pyrophosphate buffer pH 8.3, crystalline catalase (equivalent to about 100 units) dissolved in 0.2 M Tris buffer pH 8.3, an amount of enzyme to give 80-150 ~1. 02 uptake in 30 min., and water to a final volume of 3.0 ml. Different amounts of substrate (30 pmoles n-amino acids, 60 pmoles m-amino acids, 300 pmoles glycine) were put in the side arm. KOH was routinely present in the center cup. The assembled manometers were gassed with oxygen for 5 min. and then shaken at 35°C. After 34 min. equilibration, the reaction was started by adding the substrate from the side arm. After a further 5 min. of shaking, the first reading was made. Subsequent readings were taken at 5-min. intervals for 30 min. All the experiments were run in duplicate. As a measure of purification, an arbitrary enzyme unit was used which is defined as the amount of enzyme which gives 1 ~1. oxygen uptake in 30 min. Specific activity was calculated as units/mg. protein. The protein concentration was determined spectrophotometrically according to Kalckar (4). Catalase was crystallized from beef liver according to Mosimann (5). Its activity was determined with the method of von Euler and Josephson (6). Ammonia was estimated by nesslerization after steam distillation; cY-ketoglutaric acid and pyruvic acid were determined calorimetrically according to Friedmann and Haugen (7). Further identification of the keto acids was made by paper chromatography (8-10) of their phenylhydrazones prepared according to Cavallini and Frontali (11). Oxalacetic acid, when present, was determined as pyruvic acid after decarboxylation with nickel sulfate.
Puri$cation
of o-Glutamic
Acid Oxidase
The hepatopancreas of Octopus vulgaris were collected over a period of 2-3 months and kept frozen at -20°C. The organs were homogenized in a Waring blendor with cold acetone (- 20°C.) ; the suspension was filtered in a Biichner funnel and washed with cold acetone until the effluent liquid was colorless. From 10 to 15 vol. acetone was used per weight of organ. The acetone powder was dried at room temperature and kept at 4°C. in a desiccator over CaClz for no longer than 2 weeks (Table I). Step 1. The powder was carefully suspended in 15-20 vol. of 0.05 M pyrophosphate buffer pH 8.3 and extracted for 1 hr. with continuous stirring over a magnetic stirrer. This and all further operations were
338
ROCCA
AND
GHIRETTI
TABLE I f’uri$cation
of D-Glutamic
Acid.
Oxidase
5 K.-of acetone lwder) -(2 -
step -
Vol. of solution
Total units
420 220
82,000 28,600
-~~mg./ml. 67.5 4.25
220 34
22,400 12,800
1.25 1.37 0.04
ml.
1. Aqueousextract
2. First (NH&SO, precipitate 3. Acid precipitate 4. Second (NH1)804 precipitate 5. Ca gel treatment
-
10
-
1,230
70 2.89 30.4
10.5
35
81 274
28 95
27 15.6
3200
1100
1.6
made at 4°C. The suspensionwas centrifuged at 2000 X g for 30 min., and the solid material was discarded. The aqueous extract was found to be much more stable than the acetone powder; it can be kept frozen for 2-3 months without any lossof the activity. Specific activity was 2.9. Step 6. First precipitation with (NH&S04. For each 100 ml. of crude extract 14.4 g. of solid (NH&SOI was added very slowly and, after 30 min. of continuous stirring, the precipitate formed was centrifuged off. A further 11 g. of (NH,),S04/100 ml. was added to the clear supernatant, and the precipitate, which contains the enzyme, was collected by centrifugation, dissolved in 0.1 M pyrophosphate pH 8.3 and dialyzed for 12 hr. against 0.01 M Na2HP04 plus 10e3M ethylenediamine tetraacetate (EDTA) (Versene). Specific activity, 30.4; yield, 35%. Step S. Precipitation at pH 4.8. The liquid was brought to pH 4.8 with 2 N acetic acid and immediately centrifuged at 3000 X g for 15 min. The precipitate was discarded and the supernatant neutralized with 1 N NaOH. Specific activity, 81; yield, 27%. Step 4. Second precipitation with (NH&S04 . To the neutralized supernatant, 17.6 g. (NH&SO&O0 ml. was added slowly and the precipitate was centrifuged off. To the supernatant, a further 11 g./lOO ml. was added. The precipitate was collected by centrifugation, dissolved in 0.1 M pyrophosphate buffer pH 8.3, and dialyzed against 0.01 M NazHP04 plus EDTA for 12 hr. Specific activity, 274; yield, 15.6 %. This preparation was used for most of the experiments. Step 5. Treatment with calcium phosphate gel. The solution obtained in Step 4 was brought to pH 6.8 by dialyzing for 12 hr. against
D-GLUTAMIC
ACID
339
OXIDASE
1O-3 M phosphate buffer, then treated with calcium phosphate gel (0.0317 g./ml.). To each 20 ml. extract, 0.3 ml. Ca gel was added, and after 20 min. the sediment was centrifuged off. This treatment was repeated twice. The clear supernatant was then stirred with a further 2.0 ml. Ca gel which adsorbed entirely the enzyme. After washing with 1O-3 M phosphate buffer pH 6.8, the enzyme was eluted with 1O-2 M of the same buffer at pH 6.8. Specific activity, 3200; yield, 1.6 %. Properties
of D-Glutamic Acid Oxidase
Stoichiometry of the Reaction. In the presence of catalase, 1 mole of oxygen is required for the oxidation of 2 moles of n-amino acid. The relationship between oxygen uptake and formation of ammonia and keto acids was studied during the oxidation of n-glutamic acid and of n-aspartic acid. Warburg vessels with two side arms were used. Readings were taken immediately after addition of the substrate. After a certain period the reaction was stopped with 0.3 ml. of 60% perchloric acid. The contents of the vessels were cooled in ice for 10 min., then filtered, and ammonia and keto acids were determined in the deproteinized fluid. The data shown in Table II are in quite good agreement with the theoretical values. Further identification of the keto acids was obtained by paper chromatographic separation of the hydrazones formed (Table III). The oxidstion product of n-aspartic acid, viz., oxalacetic acid, was identified as pyruvic acid after treatment with 0.1 M (final concn.) ?XK~, TABLE II Stoichiometry of the Reaction The vessels contained: 1 ml. purified enzyme (1.0-1.5 mg. protein); cat&se 100 units; 0.3 ml. of 0.1 M pyrophosphate buffer pH 8.3; 30 rmoles substrate; and 0.1 ml. KOH in the center well. The final volume was made up to 3.1 ml. with water. Gas phase, oxygen. Temp., 35°C. Time of the experiment 60 min.
Substrate
n-Glutamic n-Aspartic
acid acida
0% uptake
NHa. productmn
______ ~moles
pmoles
12.1 11.2
23.7 24.2
Keto acid production a-Kg. ~___ pnmles
- 02 NH3
K-acid NH0
K-acid 02
1.08 1.06
2.12 2.31
Pyr. -/moles
25.6 25.9
0.51 0.46
a The oxalacetic acid formed was determined as pyruvic 0.1 M (final concn.) NiSO, for 30 min. at room temperature.
after treatment
with
340
ROCCA
AND
GHIRETTI
TABLE III Chromatographic Identijkation of the Keto Acids in the Reaction Fluid after Oxidation of D-Gktamic and n-Aspartic Acids by n-Glutamic Oxidase The reaction mixture was deproteinized at 0°C. with 6% (final concn.) HClO, and filtered; the keto acids were converted in phenylhydraeones and extracted according to Cavallini and Frontali (12). When n-aspartate was the substrate, oxalacetic acid was decarboxylated with NiSO, . Whatman paper No. 1. Temp., Paper
27T.
-
Reaction
a-Kg.
Method
Pyr. D-Glut.
_Rf
Ascending chromatography. 12 hr. butanolEtOH-NH3 (10:20:0.5 N) El Hawary (8). Descending chromatography. 4 hr. Paper washed with 0.1 M phosphate buffer pH 7.2 pet.rol. ether-EtOH (20:80) Kun (9). Descending chromatography. 13 hr. Chamber saturated with EtOH 95%. EtOH-water (83:17) Kun (9). Ascending chromatography. 4 hr. Chamber saturated with phenol. glycineNaOH 0.1 M pH 8.4 Virtanen (10).
RJ
0.03
0.47 0.69 0.17 0.40
0.50 0.39
0.13
RJ
-
mixture
-
n-Asp. RJ
0.03
0.20 0.47 0.05 0.17
0.73 0.82
0.51
0.55 0.70
0.44
0.39
0.45
-
0.13
-
TABLE IV of Amino Acids by Crude Extracts of Octopus Hepatopancreas The vessels contained: 1 ml. crude extract; 0.3 ml. 0.1 M pyrophosphate buffer pH 8.3; 0.3 ml. substrate (30 pmoles n-amino acids, 60 pmoles m-amino acids, 300 pmoles glycine); 0.1 ml. 15% KOH in the center well. The final volume was made up to 3.1 ml. with water. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 60 min. Oxidation
Relative
Substrate
n-Glutamate n-Aspartate Glycine n-Isoleucine nn-Isoleucine nn-Alanine n-Asparagine nn-or-Aminobutyric n-Glutamate L-Aspartate
acid
activity
100 65 43 31 31 67 74 171 0 0
D-GLUTAMIC
ACID
341
OXIDASE
Enzyme Specificity. The crude extract (Step 1) was found active on a number of o-amino acids and on glycine. L-Amino acids were not oxidized (Table IV). The activity on glycine was lost after the first (NH&SO, precipitation (Step 2). After treatment at pH 4.8, only the activity on u-glutamate and to a lesser degree on u-aspartate was retained. Neither FAD nor FMN addition was able to restore the activity on the other amino acids. This demonstrates that D-glutamic acid oxidase is not identical with the mammal D-amino acid oxidase. The ratio of activity on n-aspartate to activity on D-glutamate was also investigated. It remained constant during the purification procedure. This is an indication that the oxidation df the two amino acids is catalyzed by the same enzyme (Table V). Effect of Enzyme Concentration. The activity of the enzyme toward I,-glutamic acid was found to be proportional to the amount of protein used, when the substrate concentration was maintained constant (Fig. 1). Eflect of Substrate Concentration. The oxygen uptake was determined for various concentrations of n-glutamic and u-aspnrtic acids at pH 8.3 and 35°C. The results, plotted by the method of Lineweaver and Rurk, are represented in Fig. 2. The Michaelis constant is 8 X 1OP Al and 4.5 X lo-” ill for r)-glutamic acid and u-aspartic acid, respectively. TABLE
of
Activity
D-Glutamic
V
Acid Oxidase on n-Aspartic Acid Various Degrees of Purification
and
D-Glutarnic
Acid
at
The reaction mixture contained: 0.5 ml. extract; 0.3 ml. of 0.1 M pyrophosphate buffer pH 8.3; 0.3 ml. substrate (30 pmoles), urater to a final volume of 3.0 ml.; 0.1 ml. KOH 15yo in the center well. The oxygen uptake was followed for 30 min. Gas phase, oxygen. Temp., 35°C. ___Experiment
Iio.
step
D-Aspartic
@l. 02/30
I
II
ac.
D-Glutamic
ac.
B
A ?nin
pl. 01j30
A F
min
1 2 3 4
77.7 121 .o 187.0 239.0
120.0 159.6 152.4 304.0
0.65 0.75 0.70 0.78
1 2 3 4
31.4 53.2 30.2 66.0
61.0 80.0 23.6 100.0
0.51 0.67 0.68 0.66
342
ROCCA
AND
GHIRETTI
E$ect of pH. pH and buffer markedly affected the rates of oxidation of n-glutamic and n-aspartic acids. In Table VI the rates obtained in different buffers are reported. The strong inhibition by Verona1 was further investigated and compared with chemically related compounds (see below). The pH optimum was investigated using pyrophosphate buffer. In acid solution the activity of the enzyme decreases rapidly. n-Glutamic acid and n-aspartic acid have similar pH curves with the same maximum: 8.1-8.3 (Fig. 3). Coupled Oxa’dation with Ethyl Alcohol. Keilin and Hartree (12) showed that an oxidase system which forms hydrogen peroxide can be coupled with the oxidation of other substances such as ethyl alcohol. When the oxidation of an amino acid is carried out in the presence of EtOH, the rate of oxygen uptake is doubled. The oxidation of n-glutamic acid and
I
I
20 -
FIG. 1. Effect of enzyme concentration on the oxidation of n-glutrtmic acid. The Warburg vessel contained: different amounts of purified enzyme (Step 4); 1 ml. of 0.1 M pyrophosphate buffer, pH 8.3; catalase 100 units; 30 amoles Dglutamic acid; and water to a final volume of 3.0 ml.; 0.1 ml. KOH 15% in the center well. Gas phase, oxygen. Temp., 35°C. Ordinate, pl. oxygen uptake in 30 min. Abscissa, mg. protein.
D-GLUTAMIC I
,
,
,
,
I
,
1
ACID
343
OXIDASE
,
70 50 5c 4c JO 20 10 1
I
I
I
,
I
I
I
I
I
500
1
I
I
I
I
I
1
250
th FIG. 2a. FIG. 2. Velocity of oxidation of n-glutamic acid n-glutamic acid oxidase at pH 8.3 and temp. 35%. l/v of oxygen uptake expressed in ~1. 02 for 15 min.; l/S centration of n-glutamic acid expressed in moles/l. aspartic acid.
[sj FIG.
I
I
50‘0 .
2b.
and of n-aspartic acid by is the reciprocal of the rate is the reciprocal of the con(a) n-glutamic acid. (b) D-
TABLE VI on D-Ghtamic Acid Oxidase The incubation mixture contained: 0.3 ml. enzyme extract (Step 4) = 0.27 mg. protein; 0.3 ml. n-glutamate (30 pmoles) ; catalase 100 units; 1 ml. of 0.1 M buffer pH 8.3; and water to a final volume of 3.0 ml. Gas phase, oxygen. Temp., 35°C. Effect
.-I_
of Buffers
BUffW
Pyrophosphate Trisa Borate Ammediolb Verona1 a Tris(hydroxymethyl)aminomethane. * 2-Amino-2-methyl-l, 3-propanediol.
~1. O&Z0 min.
87.0 105.0 190.5 97.0 12.9
Relative
activity 100
121 115 112 15
344
ROCCA
AND
GHIRETTI
of nn-cr-aminobutyric acid by crude extract of octopus hepatopancreas was studied with and without a definite amount of EtOH. With eit,her substrate, the rate of oxygen uptake was doubled in the presence of EtOH. Inhibition by Benxoate. Klein and Kamin (13) first noticed that sodium benzoate in 1O-3 M concentration completely inhibits the Damino acid oxidase from mammals. We found that, whereas benzoate inhibited by 33 % the residual n-amino acid oxidase present in the crude extract of octopus hepa~pancreas, it had no effect on the purified n-glutamic enzyme. Inhibitian by Verona1 and Related Compounds. Verona1 was found to have a strong inhibitory effect on the oxidase system. Of the other relate compounds tested, only urethan inhibited (Table VII). Urea and barbituric acid were not effective. It may be remembered that Verona1 buffers are known to inhibit the activity of glycine oxidase almost completely (14).
I 80-
I
I / “\
%
$40 -T *+Oi / 20- o 0 ’ 6.0 FIG.
30,.
8 x0 FIG.
I 8.0
I.
I
8 9.0
t a0
3b.
3. Oxidation of n-glutamic acid and of n-aspartic acid by n-glutamic acid oxidase as a function of pH. All the experiments were made with 0.3 ml. purified enzyme (Step 4); 1.5 ml. of 0.1 M pyrophosphate buffer; 100 units of catdase; 30 pmoles substrate; and water to a final volume of 3.0 ml.; 0.1 ml. KOH 15% in the center well. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 30 min. The pH’s were controlled at the end of the experiment. (a) n-glutamic acid. (5) n-aspartic acid. FIG.
ph
D-GLUTAMIC
ACID
TABLE Effect
of
Sodium
345
OXIDASE
VII
Diethylmalonylurea (Veronal), Malonylurea on the D-Glutamic
Urea, Urethan Acid Oxidase
and
Sodium
The reaction mixture contained: purified enzyme Step 4 (0.3 mg. protein); 0.03 M (final concn.) pyrophosphate buffer pH 8.3; 30 pmoles n-glutamic acid; catalase 100 units. Total volume 3.0 ml.; KOH 15yo 0.1 ml. in the center well. Gas phase, oxygen. Temp., 35°C. Inhibitor
02 uptake
Concentration M
None Verona1 Verona1 Verona1 None Urea Urethane Na malonylurea
p1./30 min.
1.6 X 1O-3 8.0 X 1O-3 1.6 X 1OP 1.0 1.0 1.0
x x x
10-Z lo-2 10-z
91.7 74.2 36.6 18.8 73.7 78.7 50.8 88.0
Inhibition %
19 60 75 0 31 0
Inhibition by Suljhydryl Reagents. The importance of -SH groups for the full activity of mammal n-amino acid oxidase has been well established a long time (15, 16). It was also found that complete reversal of inhibition was produced by addition of glutathione. Similarly, n-glutamic acid oxidase was strongly inhibited by monoiodoacetat’e, o-iodosobenzoate, and p-chloromercuribenzoate. The latter showed the strongest inhibitory action: 74% inhibition with 1O-5 M. Twenty equivalents of cysteine produced complete reversal of inhibition (Table VIII). Inhibition by L-Glutamic Acid. As previously stated, L-amino acids are not oxidized either by the crude extract or by the purified enzyme. Moreover, it was found that L-glutamic acid inhibits the oxidation of n-glutamic acid. The nature of this inhibition was investigated using varying amounts of substrate and of inhibitor. The results, shown in Fig. 4, indicate the competitive nature of the inhibition. Characterization of the Prosthetic Group. The spectrophotometric study of the purified enzyme did not give satisfactory results. Attempts to resolve the enzyme into apoenzyme and prosthetic group by the method described by Warburg and Christian (17) failed. Better results were obtained with prolonged dialysis against acid (KH&SO~ . The buffer used was citrate-phosphate containing 24.3 % (NH&S04 . The pH value of the solution appeared to be critical. Treatment for 2-3 hr. at pH 3.8 gave only partial inactivation of the enzyme. In all cases the
346
ROCCA
AND
GHIRETTI
TABLE VIII of -SH Reagents and Reactivation with Cysteine The reaction mixture contained: purified enzyme 0.58 mg. protein (Step 4); 0.03 M (final concn.) pyrophosphate buffer pH 8.3; catalase 100 units; 30 rmoles n-glutamic acid. Total volume 3.0 ml. with water; KOH 15yo 0.1 ml. in the center well. Gas phase, oxygen. Temp., 35°C. Time of the experiment, 30 min. Inhibition
Inhibitor
Inhibition %
Monoiodoacetate
lXWBM 1 X 10-b M 1X lO-‘M 1 X 10-4 M plus cysteine 2 X 10-z M
p-Chloromercuribenzoate
1 1 1 1
o-Iodosobenzoate
0 48 68 18
W6 M lo-6 M lO-‘M W4 M plus cysteine 2 X W2 M
27 74 100
lXlO+M 1 X 1P M 1X10-‘M 1 x 10-a M 1 X lOma M plus cysteine 2 X 1P M
8 21 32 52
X X x X
0
0
was restored by addition of FAD. Longer treatment at, the same pH gave greater but irreversible inactivation. Treatment of the enzyme at, lower pH (2.8) for 3 hr. produced in most casescomplete loss of activity (Table IX). Neither FMN nor riboflavine were found to be able to replace FAD for the reactivation of n-glutamic acid oxidase. activity
COMMENTS
The results of the experiments described in this paper demonstrate that the enzyme purified from the hepatopancreas of Octopus vulgaris is a specific oxidase for n-glutamic acid. The identity of n-glutamic enzyme with the mammalian n-a,mino acid oxidase can be excluded since this enzyme does not attack n-glutamic acid and since the two oxidases that are present in octopus hepatopancreas can be easily separated during the purification procedure. In addition to this, sodium benzoate, which is known to be a specific inhibitor for the mammal n-amino acid oxidase, has no effect, on the n-glutamic enzyme.
D-GLUTAMIC
ACID
OXIDASE
FIN. 4. Competitive inhibition of n-glutamic acid oxidation acid. l/v represents the reciprocal of the rate of oxygen uptake oxygen/l5 min.; l/S represents the reciprocal of the concentration acid expressed in molarity. 1. No L-glutamate present. K, = 6 X 10e3M. 2. 0.1 M n-glutamate. K, = 11 X 1OP M. 3. 0.2 M L-glutamate. K, = 19.5 X lo+ M. The system contained also: 0.03 M (final concn.) pyrophosphate cntalase 100 units; different amounts of n-glutamic acid; and volume of 3.0 ml. Gas phase, oxygen; temp., 35°C.
347
by L-glutamic expressed in ml. of o-glutamic
buffer pH 8.3; water to a final
n-Glutamic acid oxidase is also active, but at a slower rate, on n-aspartic acid. That the oxidation of the two dicarboxy-u-amino acids is catalyzed by the same enzyme is demonstrated by the oxidation ratio for the two substrates, which remains constant during the purification. Green et al. (18) prepared from the “cyclophorase” system of mammals an enzyme specific for u-aspartic acid. This enzyme has in common
348
ROCCA
AND
GHIRETTI
TABLE Reactivation
with
FAD
IX
of u-Glutantic
Acid
Oxidase
after
Acid
l’reatment
The purified enzyme (Step 4) was dialyzed in the cold (4°C.) against 50 vol. of 0.05 M citrate and NazHPOI buffers containing 24.3% (NH&SO* . The precipitate was collected by centrifu~ation at 4000 X g for 5 min., washed, and redissolved in 0.1 M pyrophosphate buffer pH 8.3. The reaction mixture contained: enzyme 0.5 ml.; 1.0 ml. of 0.1 M pyrophosphate buffer pH 8.3; catalase 100 units; 0.3 ml. of 10-d M FAD; 30 pmoles n-glutamio acid. Final volume 3.0 ml. with water, 0.1 ml. KOH 15% in the center well. The readings were taken after 20 min. of incubation. Gas phase, oxygen. Temp., 35°C. Acid Enzyme
treatment
pl. Oa/30 min.
preparation
IV-2 V-l V-l VI-2 VIII-1
PH
hr.
No FAD
3.8 3.8 3.8 2.8 3.8
2 3 8 2 2
40.6 45.0 9.3 5.4 66.5
With
FAD -
76.0 80.5 36.8 26.0 136.7
with the octopus n-glutamic enzyme the insensitivity to benzoic acid, but differs from it with respect to its substrate specificity. As for the inhibitory action of Verona1 and urethan, it must be remembered that Verona1 inhibits also the activity of glycine oxidase almost complexly (14). The experiments of reactivation after acid treatment indicate that n-glutamic acid oxidase from octopus is a flavoprotein. Flavoproteins are known to produce hydrogen peroxide when reacting with molecular oxygen. It follows therefore that the ratio of oxygen uptake in the absence and in the presence of catalase is 2: 1. The relatio~~p between oxygen uptake and substrate oxidized when excess of catalase was present, demonstrated that hydrogen peroxide is produced during the oxidase reaction. The formation of peroxide was further demonstrated by the possibi~ty of coupling the oxidative deamination of n-glutamate or n-aspartate with the oxidation of ethyl alcohol. Under the conditions used in this study, n-glutamic acid oxidase could be partly resolved into apoenzyme and prosthetic group; reactivation occurred when the dialyzed preparation was supplemented with FAD. Other prosthetic groups like F&IN or riboflavine cannot replace FAD. The difficulty with which the n-glutamic enzyme is split under acid conditions, compared with the relative mild conditions employed for the mammal n-amino acid oxidase and the n-aspartic
D-GLUTAMIC
340
ACID OXIDASE
acid oxidase, can be regarded as evidence that in octopus enzyme the prosthetic group is more firmly bound to the npoenzyme.
The authors script.
wish
to thank
Prof.
8. M.
Rapoport
who
kindly
revised
the manu-
SUMMARY 1. A procedure is described for the purification of wglutamic acid oxidase from the hepatopancrens of Octopus vulgaris. 2. Optimum conditions are given for the assay of the enzyme. The activity is higher in oxygen than in air. The influence of buffers, of enzyme concentration, and of substrate concentration is studied. The ratio of oxidation for n-glutamic and n-aspartic acids is presented together with the relative rates of oxidation of other amino acids. 3. The nature of the prosthetic group has been investigated. The experiments of reactivation after acid treatment indicate that the enzyme is a flavoprotein with flavine adenine dinucleotide as the prosthetic group.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. !). 10. 11. 12. 13. 14. 15. 16. 17. 18.
BLASCHKO, H., AND HAWKINS, J., Biochem. J. 62, 306 (1952). BLASCHKO, H., AND HIMMS, J. M., J. Physiol. (London) 128, 7P (1955). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) Bl24, 397 (1938). KALCKAR, H. N., J. Biol. Chem. 167, 461 (1947). MOSIMANN, W., Arch. Biochem. Biophys. 33, 487 (1951). EULER, H. VON, AND JOSEPHSON, K., Ann. 462, 158 (1927). FRIEDMANN, E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). EL HAWARY, F. S., AND THOMPSON, R. H. S., Biochem. J. 63, 34 (1953). KUN, E., AND GARCIA-HERNANDEZ, M., Biochim. et Biophys. Acta 23, 181 (1957). VIRTANEN, A. I., MIETTININEN, J. K., AND KUNTNER, H., Beta Chem. Scar&. 7, 38 (1953). CAVALLIXI, D., AND FRONTALI, M., Biochim. et Biophys. Acta 13, 459 (1953). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) B119, 114 (1936). KLEIN, J. It., AND KAMIN, H., J. Biol. Chem. 138, 507 (1941). RATNER, S., NOCITO, V., AND GREEN, D. E., J. Biol. Chem. 162, 119 (1944). SINGER, T., AND BARRON, E. S. G., J. Biol. Chem. 167, 241 (1945). SINGER, T., J. Biol. Chem. 174, 11 (1948). WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 298, 150 (1938). STILL, S., BOELL, M. V., KNOX, W. E., AND GREEN, D. E., J. Biol. Chem. 179, 831 (1949).