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CXLI. GLUTAMIC ACID DEHYDROGENASE
FROM GERMINATING SEEDS'
By MANAYATH DAMODARAN AND KESAVAPILLAY RAMAKRISHNAN NAIR
From the Biochemical Laboratory, University of Madras, Madras
(Received 23 March 1938)
LITTLE definite knowledge exists as to the mechanism of oxidative deamination in plants. That such deamination does occur is to be inferred both from the values of the respiratory quotients of certain germinating seeds [Malhotra, 1933] which indicate protein catabolism, as well as from the well-known accumulation of asparagine and^ glutamine during germination which^ has been shown^ to^ take place only in the presence of oxygen [Palladin, 1889]. As the amount of aspara- gine found is usually far^ in^ excess^ of^ what^ can^ be^ accounted for^ on^ the^ basis of the aspartic acid content of the seed proteins and, further, as the increase in asparagine is^ concomitant with the^ disappearance^ of^ other^ amino-acids^ [Schulze, 1898; 1901] there can be no doubt that its formation takes place at the expense of the latter. It is generally assumed that the ammonia formed by oxidative
deamination condenses with COOR.CO.CH2.GCOOH arising either^ from carbo-
hydrates or from deaminized amino-acid residues to give first aspartic acid and then its amide. In the case of glutamine the^ position is^ rather^ different^ on account of the high content of the seed proteins in glutamic acid. As Greenhill & Chibnall [1934] have pointed out, with^ the^ evidence^ at^ present available^ it is equally valid to assume either that the glutamine present in the seed is the direct product of the^ hydrolysis of^ protein^ [Damodaran^ et^ al.^ 1932]^ or^ that^ it arises from the combination of ammonia with a non-nitrogenous residue. One of^ the^ most^ interesting results of^ recent^ work^ on^ the^ oxidation^ of^ amino-
acids in animal tissues is the hypothesis due to Krebs [1935, 1, 2] that in kidney
tissue glutamine synthesis takes place through the intervention ofa "^ deaminase ". The disappearance of ammonia during the oxidation of glutamic and aspartic acids by muscle was noted by Needham (^) [1930). Krebs observed a similar effect with glutamic acid in the presence of kidney slices; he was able to show that
the disappearance of ammonia was accompanied by an increase of amide-N
determined by the method of Sachsse^ [1873]^ and further that^ the^ disappearance
of added ammonia could be brougbt about by kidney slices in the presence of
oc-ketoglutaric acid. These observations taken^ in^ conjunction with the^ existence of a glutaminase in kidney have led Krebs to the inference that in the intact
organ the^ deaminase system studied^ by him^ is^ really concerned, not^ with^ the
oxidation of glutamic acid, but with the reverse synthetic process, viz. the
formation of glutamine from^ ammonia^ and^ ketoglutaric acid,^ the^ latter^ probably
arising from carbohydrate oxidation via pyruvic acid and omoc-diketoadipic acid
[Toenniesen &^ Brinkmann, 1930].
It is of obvious interest to ascertain if^ this^ very attractive^ hypothesis is
applicable to the vegetable organism also, especially in view of the fact that the
experimental evidence for the formation of^ glutamine adduced^ by Krebs^ rests
First appeared as a note^ in^ Curr.^ Sci.^ (1936), 5, 134. ( 1064 )
GLUTAMIC ACID DEHYDROGENASE
solely upon the determination of amide-N by acid hydrolysis, a method which can hardly be called specific [cf. Chibnall & Westall, 1932], whereas the formation
of glutamine in the plant has been proved beyond doubt by actual isolation by
a number of workers. The present paper describes in the first place experiments carried out to ascertain the existence of deaminases in germinating seeds and secondly the properties of the only amino-acid dehydrogenase^ which^ was actually found^ in the seedlings examined, viz. one specific for glutamic acid. Maceration extracts of the seedlings of about a dozen species smostly of the leguminous family) were tested, by the methylene blue and the manometric methods, for their actions on the^ following^ amino-acids: glycine, alanine,^ leucine, tyrosine, histidine, aspartic acid, glutamic acid and dl-proline. Glutamic acid was the only amino-acid^ that^ was acted^ upon and even this action was found to be limited to three out of the twelve species examined, viz. to Phaseolus mungo, P. radiatus and Pisum sativum. In being obtained in cell-free extracts (^) capable of oxidizing the natural l(+)glutamic acid the enzyme found in germinating seeds differs from the deaminases studied by Bernheim and coworkers [1934, 1, (^) 2; 1935, 1, 2] by Krebs [1933, 1, 2; 1934; 1935, 1, 2, 3] and by Weil-Malherbe [1935; 1936] whose preparations in extracts free from cellular material acted only on the optically non-natural amino-acids. Purification^ of the^ dehydrogenase by adsorption on kaolin, kieselguhr and aluminium hydroxide proved un- successful. Some concentration could be^ effected^ by^ precipitation^ of the^ macera- tion extract by saturation with ammonium sulphate. But the precipitate thus obtained could not^ be^ freed^ from^ ammonium sulphate as dehydrogenase activity was completely lost during dialysis. For all experiments, therefore,^ the^ am- monium sulphate precipitate redissolved in phosphate buffer at pH 7-8 and without dialysis was used. Seedlings of Phaseolus (^) mungo two days old were found (^) to be the best starting material. The initial oxygen uptakes of such preparations were quite low being generally about 50 (^) pl./hr./ml. The enzyme was always contaminated with peroxidase. It could, however, be readily demonstrated by means of differential inactivation that peroxidase activity was in no (^) way responsible for the oxidation of glutamic acid. Thus treatment with
acetone had very little influence on peroxidase (as determined quantitatively by
the oxidation of Nadi reagent) while glutamic acid dehydrogenase was com- pletely destroyed by the process.
Being capable of utilizing either oxygen or methylene blue as hydrogen
acceptor the enzyme belongs to the class of^ oxytropic^ dehydrogenases.^ The action in the presence of oxygen is not inhibited by KCN up to a concentration
of 0*005M or by the other^ inhibitors^ tried, viz.^ arsenite^ and^ fluoride.^ The
following substances were tried on the enzyme system to determine if they could function as hydrogen transporters^ in the^ reaction: methylene^ blue,^ ascorbic^ acid, glutathione and dihydroxyphenylalanine. None of these were found to have any accelerating effect on the oxygen^ consumption. Inactivation by alcohol precluded the use of the method based on the coupled oxidation of alcohol by which Keilin & Hartree [1936] had demonstrated the formation of hydrogen peroxide in the case of kidney deaminase. The "Abfangsverfahren" of Wieland & Rosenfeld [1930] using ceric sulphate also gave negative results. But taking advantage of the presence of^ peroxidase^ in^ the enzyme preparation used, productionof hydrogen peroxide could be demonstrated by the coupled oxidation of nitrite [Thurlow, 1925]. The oxygen uptake corresponds to one atom of oxygen to each molecule of glutamic acid. The end product of the reaction was shown^ to be^ oc-ketoglutaric
lOff
GLUTAMIC ACID DEHYDROGENASE
Table I. Time for decoloration of methylene blue in Thunberg tubes (Figures in brackets refer to substrate-free controls. In boiled controls the time was the same in all cases.) Time in minutes
Seedling tried Phaseolu8 mungo
Pha8eolus radiatws
Dolichos lab lab
Dolichos biflorus Cicer arietinum
Canavalia ensiformi
Canavalia obtusifolia
Pisum sativum
CitriUus (^) colocynthis
Coconut Wheat CitriUus (^) ulgaris
Glycine 23 (23) 12 (12) 22 (22)
6 (6) 35 (35) 31 (30) 22 (22) 82 (85)
Alanine 17 (21) 10 (12) 25 (25)
6 (6) 27 (27) 33 (33) 18 (19) 78 (76)
Tyro- Histi-^ Aspartic sine dine acid Leucine 28 28 19 23 (28) (28) (19) (24) 12 16 13 9 (12) (16) (12) (10) 25 30 28 23 (25) (29) (28) (23) No decoloration in 2 hr. 8 ( 27 ( 41 ( 23 ( 82 (
8 9 8 (8) (8) ( 32 33 30 (33) (33)^ ( 41 37 36 (41) (36) ( 22 19 19 (22) (19) ( 76 78 81 (76) (76) ( No decoloration in 2 hr. No decoloration in 2 hr. No (^) decoloration in 2 hr.
Glutamic dl- acid Proline 6 22 (18) (22) 5 13 (11) (12) 23 23 (23) (23)
5 6 (6) (6) 24 26 (25) (25) 32 38 (33) (36) 8 21 (19) (22) 80 69 (82) (69)
Purification of the enzyme Precipitation with dilute acetic acid, sodium sulphate and acetone and electrodialysis were tried as methods of purification, but all the methods yielded inactive preparations. The enzyme was not adsorbed on kaolin, kieselguhr or aluminium hydroxide. Precipitation by saturation with ammonium sulphate and taking up in phosphate buffer at pH 7-8 yielded an active preparation^ with a low initial oxygen uptake. Seedlings between 2 and 3 days' old yielded the most active preparation. 20 ml. of the extract (pH 6-1) were cooled in ice and brought to pH 5-2 by the addition of very dilute acetic acid.^ After^ saturation with^ ammonium^ sulphate
and centrifuging for 10 min. at 2000 r.p.m. the clear supernatant liquid was
discarded. The residue was washed with phosphate buffer at pH 5-2 and re- dissolved in phosphate buffer at pH 7-8. The preparation in the absence of glutamic acid did not appreciably reduce methylene blue and had an oxygen
uptake of about 50 ,ul./hr./ml.
Differentiation from peroxidase activity Peroxidase was invariably found in the seedlings and persisted in the pre- paration obtained as described above. Previous attempts at purification had shown that drying with acetone destroyed dehydrogenase activity.^ A^ com- parison was therefore made of the effects of acetone treatment on the peroxidase activity and on the oxidation of^ glutamic^ acid. 20 g. (^) of the seedlings were extracted with 20 ml. 0-87 % K2HPO4- The extract was poured slowly with stirring into five times the volume of acetone cooled in ice. The precipitate was washed on the centrifuge with 5 ml. of acetone, spread out on a Petri dish and the acetone driven off by aeration. The yellowish white powder so obtained was suspended in 20 ml. of phosphate buffer at^ pH^ 7-
1068 M. DAMODARAN AND K. R. NAIR
and the activities of the dehydrogenase and peroxidase determined by the mano- metric method and by Guthrie's method respectively. For comparison the same determinations were done on the fresh extract of the seedlings. The results of three different experiments are given in Table II.
Table II. Effects of acetone treatment on (^) dehydrogenase and peroxidase Peroxidase Dehydrogenase Length of column in colorimetric Oxygen uptake with glutamic acid
tube (standard at 20 mm.) (pl./hr.)
A (^) A , A
After peroxidase 26-1 76- 38.9 76* 13*1 77.
Before 238- 250- 242-
% dehydro- After genase 0 0 0 0 0 0
The results clearly indicate that the oxidation of glutamic acid is uncon- nected with peroxidase. Though more than 75 (^) % of the peroxidase remained after acetone-drying the manometric oxygen uptake was zero.
Variation of activity with pH The variation of the oxygen uptake with pH is illustrated in Fig. 1. Below
pH 5*9 the enzyme is totally inactive. The optimum pH is 7-8-8-0. The reduction
of methylene blue in Thunberg tubes does not appear to be so susceptible to changes in pH as the^ oxygen uptake (Table III); at^ pH 5 9, however, the reduction of methylene blue also ceases.
350H
300
250
200-
1501-
100
50
5-5 6-0 6-5 7-0 7-5 8- pH of medium Fig. 1.
8-5 9-0 9.
Before 18* 29* 10*
I (^) I I
I I I (^) I I I
M. DAMODARAN AND K. R. NAIR
Inactivation by heat 10 ml. of the enzyme solution were placed in each of several test tubes kept immersed for different periods in baths at different temperatures. The solutions were cooled and the dehydrogenase activity in each tested manometrically.
Slight inactivation was evident even at 370. At 500 and above the destruction
of the enzyme was very rapid (Table V).
Table V. Inactivation by heat Temperatures Time of -,A heating 370 400 500 600 700 800
min. pi.-1 ,uPi P.I1L. 91.
5 362-0 352-5 182-5 17-5 0 0 30 341-0 245-0 0 0 0 0 60 328*5 97 5^0 0 0
Formation of hydrogen peroxide Fixation of hydrogen peroxide by oxidation of^ cerium^ hydroxide to^ cerium peroxide [Wieland & Rosenfeld, 1930] and determination of the cerium peroxide
by permanganate titration^ failed^ to^ yield any clear-cut^ evidence^ of the^ formation
of hydrogen peroxide. The coupled oxidation of alcohol was also inapplicable
as the alcohol concentration used by Keilin & Hartree [1936] was found to retard the dehydrogenase action. It was found, however, that the method used by Thurlow [1925] for xanthine oxidase, based on the oxidation of nitrite to
nitrate by peroxidase at the expense of the^ hydrogen peroxide formed by
dehydrogenation, could be applied here on account of the presence of peroxidase in the enzyme preparation. The^ enzyme was^ allowed^ to^ act^ upon^ glutamic acid in the presence of added sodium nitrite, the reaction mixture being aerated
for a^ definite period. At^ the^ end^ the^ solutions^ were^ centrifuged and the^ amount
of unoxidized nitrite determined colorimetrically using Griess-Ilosvay's reagent.
Table VI gives the composition of the reaction mixture and the colorimetric readings in a typical experiment in which the time of aeration was 45 min.
Table VI. Coupled oxidation of nitrite
Flasks ... ... 1 2 3 Enzyme 10 ml.^10 ml. Glutamic acid (^) M/10 5 ml.^5 ml. Sodium nitrite^ 0X02 % 2 ml.^ 2 ml.^2 ml. Buffer pH 7-8 - ml. 10 ml. Unoxidized nitrite (as length of column in 355 mm. 6-2 mm. 5*1 mm. colorimetric tube to balance standard set at (^5) mm.)
Effect of various^ hydrogen transporters
Appropriate experiments showed that^ methylene blue, ascorbic^ acid, gluta-
thione and dihydroxyphenylalanine were incapable of acting as intermediate
carriers in the oxidation of gjutamic acid^ by the^ dehydrogenase. In^ no case^ was
a significant acceleration of oxygen uptake observed on the addition of these
substances. Inhibiting agents
KCN up to 0*005M had little effect^ on^ the^ oxidation^ (Table VII^ and^ Fig. 3).
With higher concentrations the rate fell appreciably but the oxygen consumption
was not completely inhibited^ even^ by a^ cyanide concentration^ of 0-2M.^ Arsenite
l
GLUTAMIC ACID DEHYDROGENASE
Ca 200-
150-
0 10 20 30 40 50 60 70 Time in (^) min. Fig. 3. a=without (CN); b =0 001 M (CN); c =0-002 M (CN); d=0005 M (CN); e=0 01 M (CN); f=0 1 M (CN); g=0-2M (CN).
250
200-
150-
50
(^0 10 20 30 40 50 60 70 ) Time in min.
Fig. 4. a and b-no arsenite; c=0001 M arsenite; d=0*002 M arsenite; e=0-02 M arsenite.
GLUTAMIC ACID DEHYDROGENASE 1073
Table VIII. Oxygen con8umption with 1 ml. of M/50 glutamic acid
pl. oxygen^ taken^ up
Time in mm. Exp. Control Difference 10 30 6 10.1 20- 20 457 20-5 25- 30 98-7 317 67- 40 122-6 38-9 83- 50 153-1 43-1 110. 60 1932 51.0 142- 70 216-7 61-7 155 80 2407 68-2 172- 90 260-5 735 187- 100 2853 80-1 205- 110 308-2 86-2 222 120 3200 90.0 230 130 325,5 95 5 230- 140 328-7 98-5 230-
Isolation of (^) oc-ketoglutaric acid as the end product of the oxidation To 250 ml. of the enzyme solution in M/15 phosphate buffer at pH 7-8, 100 ml. of M/10 glutamic acid neutralized with NaOH and 0 079 g. of arsenic trioxide as sodium arsenite were added and the mixture gently shaken for 2 hr. in a stoppered 500 ml. flask. It was found helpful to add 10 ml. of 1/ methylene blue to the mixture, the disappearance of the colour indicating the necessity of admitting fresh air and its permanence marking the end point of the reaction. At the end of 2 hr. the proteins were precipitated by adding 50 ml. of (^50) % trichloroacetic acid, the liquid filtered at the pump using filter (^) paper impregnated with kieselguhr and the filtrate concentrated to 150 ml. in vacuo. 100 ml. of^ a 2^ % solution of^ 2:4-dinitrophenylhydrazine^ in^ 2N^ HCI^ were^ now added and the mixture kept in the refrigerator for 12 hr. The crystalline yellow precipitate which formed was filtered and purified by dissolving in 2N NaOH
and reprecipitating with RId. The precipitate thus obtained was separated on
the centrifuge, washed several times with water and recrystallized from ethyl acetate. Golden yellow needle-shaped crystals of the dinitrophenylhydrazone
of o-ketoglutaric acid melting at 2230 were obtained. Yield 700 mg.
In a (^) control experiment without the addition of glutamic acid (^11) mg. of the same product were obtained. It is assumed that this arose probably from the glutamic acid formed in^ the^ breakdown of protein which^ was not^ completely removed by the method adopted for the purification of the enzyme.
SUMMARY
A number of germinating seeds have been examined for the presence of amito-acid dehydrogenases. The only such dehydrogenase found is one specific
for glutamic acid which occurs in the seedlings of Phaseolus mungo, P. radiatus
and Pisum sativum. The enzyme is readily obtained in aqueous extracts^ free from cellular^ material and can be concentrated by precipitation with ammonium sulphate and re- dissolving in phosphate buffer.^ In this^ condition^ it^ actively^ oxidizes^ the^ naturally
occurring l(+)glutamic acid, differing in this respect from deaminases from
animal sources hitherto studied, which in cell-free extracts act only on the
optically non-natural isomerides of the amino-acids.
The enzyme belongs to the class of aerobic dehydrogenases, being capable of
oxidizing glutamic acid either in the presence of methylene blue or of molecular
Biochem. 1938 xxxii^68
M. DAMODARAN AND K. R. NAIR
oxygen. Hydrogen peroxide formation has been demonstrated by the coupled
oxidation of nitrite. Cyanide, arsenite and^ fluoride^ do not inhibit the enzyme nor is it activated by methylene blue, ascorbic acid, glutathione or dihydroxy- phenylalanine. The oxygen uptake corresponds to 1 atom of oxygen per molecule of glutamic acid. The end product of the oxidation is a-ketoglutaric acid, which has been isolated and identified as the 2: 4-dinitrophenylhydrazone.
REFERENCES
Bernheim & Bernheim (1934, 1). J.^ biol.^ Chem.^ 106, 79. (1934, 2). J. biol. Chem. 107, 275.
- (1935, 1). J. biol. Chem. 109, 131. & Webster (1935, 2). J.^ biol. Chem. 110, 165. Chibnall & Westall (1932). Biochem. J. 26, 22. Damodaran, Jaaback & Chibnall (1932). Biochem. J. 26, 1704. Dixon (1934). Manometric methods. (Cambridge University Press.) Greenhill & Chibnall (1934). Biochem. J. 28, 1422. Guthrie (1931). J. Amer. chem. Soc.^ 53, 242. Keilin & Hartree (1936). Proc. roy. Soc. 119, 114. Krebs (1933, 1). Hoppe-Seyl. Z. 217, 191.
- (1933, 2). Hoppe-Seyl. Z. 218, 157. (1934). Hoppe-Seyl. Z. 230, 278. (1935, 1). Biochem. J. 29, 1620. (1935, 2). Biochem. J. 29, 1951. (1935, 3). Biochem. J. 29, 2077. Malhotra (1933). J. Biochem. Japan, 18, 173. Needham (1930). Biochem. J.^ 24, 208. Palladin (1889). Ber. dt8ch. bot. Gem. 7, 126. Sachsse (1873). J. prakt. Chem. 6, 118. Schulze (1898). Landw.^ Jb. 27, 503.
- (^) (1901). Landw. Jb. 30, 287. Thunberg (1920). Skand. Arch. Physiol. 40, 1. Thurlow (1925). Biochem.^ J.^ 19, 175. Toenniesen & Brinkmann (1930). Hoppe-Seyl. Z. 187, 137. Weil-Malherbe (1935). Chem. and Ind. (^) 54, 1115.
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