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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 240, No. 5, May 1965

Printed in U.S.A.

Glutamate Dehydrogenase

VI. SURVEY OF PURINE NUCLEOTIDE AND OTHER EFFECTS ON THE ENZYME FROM VARIOUS SOURCES*

CARL FRIEDEN

From the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

(Received for publication, December 28, 1964)

It is now well known that the activity of the glutamate dehydrogenase (L-glutamate:DPN (TPN) oxidoreductase (deaminat- ing), EC 1.4.1.3) from beef liver is strongly and specifically influenced by purine nucleotides, in particular by the adenosine and guanosine di- and triphosphates (2,3). Further, it has been amply demonstrated that these nucleotides bind to a specific site on the enzyme, distinct from any “active” site (2). As a consequence of such binding, these nucleotides, in conjunction with the coenzymes for the reactions, influence the molecular and kinetic properties of the enzyme, presumably as a result of a configurational change within the protein itself (2, 4, 5). At high enzyme concentrations, this configurational change is reflected in the ability of the protein to undergo a reversible association-dissociation reaction (2).

Glutamate dehydrogenases have been isolated from a large number of sources. So far, little comparative enzymology had been performed on these proteins, although such studies should be of considerable interest since there are, as will be seen, both subtle and quite marked differences between these enzymes. As a preliminary investigation, we believed it to be of some interest to investigate different glutamate dehydrogenases with respect to the effect of purine nucleotides on the enzyme activity. The investigation might serve the dual purpose of examining the role of purine nucleotide in affecting the molecular properties, that is, the configurational alteration of the protein, as well as giving information concerning the control of enzyme activity in relation to over-all metabolism. This study is aided considerably by the fact that crystalline preparations have been obtained from three different sources (beef, chicken, and frog) and that the enzyme may be purified relatively easily from a large number of other sources. A preliminary report has already indicated distinct differences in these enzymes (1).

EXPERIMENTAL PROCEDURE

Materials-Coenzymes and purine nucleotides used were obtained from the Sigma Chemical Company, St. Louis. Crys- talline beef liver glutamate dehydrogenase was obtained from C. F. Boehringer and Sons, Mannheim, Germany, either as a suspension in (NHJzS04 or in 50% glycerol. In most cases, these two preparations appear to be identical under the conditions used in thii paper. It was noted, however, that the glycerol enzyme was sometimes less active and ultracentrifugation

* A preliminary report of this investigation has been presented (1). This work was supported by Research Grant GM 08117 from the United States Public Health Service.

of this material showed, in addition to the usual enzyme peak, a slower moving peak which apparently is inactive protein. When the glycerol is removed (by Sephadex, for example), the slow moving peak is no longer present, presumably due to the fact that the inactive protein precipitates in the absence of glycerol.

Assays-All assays were performed at pH 8 in 0.01 M Tris- acetate (0.01 M with respect to acetate), 10 PM EDTA, and at 25’. The concentration of a-ketoglutarate and NH&l were 5 mM and 50 mM, respectively. Initial velocities were obtained with an expanded scale recorder (0.1 optical density full scale) as previously described (6).

Enzymes--Glutamate dehydrogenases from various sources were prepared as described below. All steps were carried out at &3” except where noted. Acetone powders, when used, were prepared by homogenizing the tissue with 5 volumes of acetone ( -10’) for 1 minute and pouring the homogenate into a mini- mum of an equal volume of acetone. The slurry is stirred for several minutes and filtered. The filter cake is taken up in 10 volumes of acetone (-lo”), stirred until smooth, and then re- filtered. The cake is then dried by hand at room temperature.

Chicken Liver-Crystalline enzyme was prepared from an acetone powder following the procedure outlined by Snoke (7). This paper contained a typographical error and 122 ml of a ribose nucleic acid solution which contains 5 g of ribose nucleic acid in 100 ml inst.ead of 50 g should be added to each liter of phosphate buffer extract.

Pigeon Liver-Pigeon liver acetone powder was obtained from the Sigma Chemical Company. The powder was extracted with 15 times its volume with 0.01 M phosphate, pH 7.4, for 1 hour. The pH of the extract was lowered to 5.25, the extract was centrifuged at 2000 x g for 10 minutes, and the supernatant fluid wss discarded. The precipitate was taken up in 0.01 M

phosphate, pH 7.2, and then fractionated with ammonium sulfate, the fraction between 25 and 40% saturated ammonium sulfate being retained. The precipitate from this fraction was dissolved in 0.1 M phosphate buffer, pH 7.2, and protamine sulfate, corresponding to about one-fortieth the amount of protein in the solution was added. The solution was then clarified and made up to 25% saturation with ammonium sulfate. After centrifugation, the supernatant fluid was brought to 35% ammonium sulfate and allowed to stand overnight. The precipitate was again fractionated with ammonium sulfate and this material was used for the kinetic studies.

Pig Kidney-Enzyme was prepared from an acetone powder

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May 1965 C. Frieden

of pig kidney. The dry powder was extracted with 10 volumes of cold 0.05 M phosphate buffer, pH 7.2. After 1 hour, the solution was centrifuged at 1000 x g for 30 minutes and the precipitate discarded. The pH of the supernatant fluid was lowered to 4.8 with 10% acetic acid and the precipitate which formed was centrifuged out. Most of the enzyme precipitated and the supernatant fluid was discarded. The precipitate was taken up in 0.1 the original volume with 0.05 M phosphate buffer, pH 7.2. This material was fractionated twice with ammonium sulfate, the first fraction being 0 to 30% and the second fraction 30 to 40%. The material from the second ammonium sulfate fractionation was stored as a suspension under 50% ammonium sulfate.

With the activity of the crystalline beef liver glutamic dehydrogenase as a criterion of purity, the kidney enzyme was approximately 20 to 30% pure after the above fractionation.

Calf Brain-Two different procedures were used in the preparation of brain glutamate dehydrogenase. In the first, brain was homogenized in 3 volumes of 0.01 M phosphate, pH 7.4, and the homogenate centrifuged and fractionated with ammonium sulfate. The fraction between 25 and 500/, was then used for the preparation of an acetone powder. This powder was then extracted with 0.1 M phosphate buffer, pH 7.2, for 1 hour, and the extract fractionated with ammonium sulfate again with the fraction which precipitated between 25 and 50% being used in kinetic experiments.

The second procedure was that reported by Grisolia, Quijada, and Fernandez (8). Homogenized brain tissue is subjected to ammonium and sodium sulfate fractionations and several heat steps. The procedure of Grisolia et cd. was modified somewhat by careful ammonium sulfate fractionation at the end, resulting in some additional purification of several fold.

Although the methods of preparation were quite different, the enzyme behaved essentially identically regardless of which preparation was tested.

Beef Heart-An acetone powder was prepared from fresh minced beef heart. The powder was extracted with 0.05 M

phosphate buffer, pH 7.4, and the pH lowered to 4.8 with 1 N

acetic acid. The supernatant fluid was discarded and the precipitate, which contained most of the enzyme, was taken up in phosphate buffer, pH 7.4. This enzyme preparation had a considerable amount of DPNH oxidase, but no TPNH oxidase and, therefore, kinetic experiments were performed with TPNH as coenzyme.

Rabbit Muscle-Frozen rabbit muscle was used to prepare an acetone powder. The dry powder was extracted with 10 volumes of 0.01 M phosphate buffer, pH 7.2, for 30 minutes and the solution was centrifuged at about 8000 x g for 10 minutes. The supernatant fluid was then fractionated with ammonium sulfate, the enzyme precipitating between 25 and 50% ammonium sulfate. The precipitate from the ammonium sulfate fractionation was turbid when taken up in phosphate buffer and no attempts were made to purify the enzyme further. The preparation was, therefore, quite crude.

Frog Liver Glutamate Dehydrogenase-Crystalline frog liver glutamate dehydrogenase was a gift of Drs. P. P. Cohen and L. Fahien. In contrast to most of the other preparations of the enzyme from animal tissues, an acetone powder was not used from the preparation of the enzyme. Instead, the enzyme was extracted from an homogenate with cetyltrimethylammonium

bromide and subsequently purified with ammonium sulfate fractionations. Enzyme, not so extensively purified, has been prepared with an acetone powder and extracted with phosphate. The extract was then fractionated with ammonium sulfate. Although this enzyme was not as pure as the other, the kinetic properties were essentially identical.

Tadpole Liver Glutamate Dehydrogenase-This enzyme was a crude preparation prepared by the same method as the frog liver enzyme (cetyltrimethylammonium bromide). The kinetic experiments were performed on enzyme kindly supplied by Dr. L. Fahien.

Trout Liver-An acetone powder was prepared from 12 g of fresh trout liver. The dry powder was extracted with 10 times its volume of 0.05 M cold phosphate buffer, pH 7.2, for 1 hour. The solution was then clarified by centrifugation at 12,000 x g for 15 minutes. This material was fractionated twice with ammonium sulfate, the first cut being between 33 to 63% saturation and the second 33 to 53y0. The activity was low and the protein concentration rather high so that this preparation is crude.

Escherichia coli (TPN-dependent Enzyme)-Escherichia coli (ATCC 53961) was grown in a medium consisting of 8 g of nutrient broth, 3 g of glucose, 3 g of yeast extract, 5 g of KaPO+ and 2 g of glutamic acid in 1 liter of solution. Cells from 2 liters of this medium were harvested and washed twice with 150 ml of 0.1 M phosphate buffer, pH 7.2. The cells were then suspended in 50 ml of the same buffer and subjected to sonic oscillation at 10 kc for 12 minutes. After sonication, the solution was centrifuged at 100,000 x g for 1 hour and the precipitate discarded. The supernatant fluid was fractionated with ammonium sulfate, with the enzyme being precipitated in the 35 to 55% fraction. This precipitate was taken up in 10 ml of 0.1 M phosphate buffer, pH 7.2, and heated at 53” for 30 minutes. The solution was cooled and the heavy precipitate spun out, Small quantities of ribonuclease and deoxyribonuclease were added and the solution was warmed to 37” for 10 minutes. The solution was cooled and fractionated with ammonium sulfate. The enzyme precipitated between 40 and 55% saturation and this precipitate was taken up in 0.1 M phosphate buffer, pH 7.2. At this stage, the enzyme had been purified lo- to 20.fold over the sonic extract. The enzyme could be further purified, about 2- or a-fold, by placing it on a DEAE-Sephadex column pre- equilibrated with 0.01 M phosphate buffer, pH 7.2, washing t,he column with this buffer, and eluting the enzyme with 0.3 M

phosphate. Enzyme purified 20- to 50-fold over the sonic extract was used for all kinetic experiments. Enzyme stored in 50% ammonium sulfate was stable for several months.

Neurospora crassa, Strain 5%‘97a (Separate DPN- and TPN- dependent Enzymes)-Spores of this strain, kindly supplied by Dr. S. Kinsky, were grown on minimal medium either with NH4NOa or urea as the nitrogen source. The DPN-dependent enzyme was purified from the material grown with urea and the TPN-dependent enzyme from the material grown with NH4N03. In both cases, enzyme was purified by the procedure described by Sanwal and Lata (9).

Corn Leaf (DPN-dependent Enzyme)-Purified glutamate dehydrogenase from corn leaves was prepared by the method of Bulen (10). The enzyme was purified about 25-fold over the crude extract and this material was used for the kinetic experiments reported.


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2030 Glutamate Dehydrogenase. VI Vol. 240, No. 5

TABLE I Enzymes Unaflected by Purine Nucleotides Xumm,ary of extents of purijication of glutamate dehydrogenases

from various sources In general, all the glutamate dehydrogenases from nonanimal

(bacterial, yeast, Neurospora, corn leaf) sources are neither inhibited nor activated by purine nucleotides at concentrations below 100 PM. It should be noted that at concentrations of approximately 1 mM or higher, the different purine nucleotides did tend to activate coenzyme oxidation. Activation was never greater than 1.5- to 1.6-fold and was similar for ADP, ATP, GDP, and GTP. Since the concentrations required were so high, it is felt that these effects represent nonspecific binding of polyanions to the enzyme.

Source

1. Beef liver. . . 2. Chicken liver.. 3. Pigeon liver. 4. Pig kidney.. . 5. Calf brain. 6. Beef heart.. . 7. Rabbit muscle. 8. Frog liver. 9. Tadpole liver.

10. Trout liver. . 11. E. coli (TPN-dependent). 12. N. crassa (DPN-dependent)

N. crassa (TPN-dependent) 13. Corn leaf (DPN-dependent) . 14. Yeast (TPN-dependent)

Purity

% 100* 100*

lot 2s30t lo-2ot

1t 0.2t

100*

l$ 0.53:

Purification over extract

-fold

20-50 30-50

140

30 100

* Crystalline and homogenous in ultracentrifuge. t Specific activity relative to the specific activity of the beef

liver glutamate dehydrogenase. $ Specific activity relative to the specific activity of the frog

liver enzyme.

TABLE II

Effects of purine nucleotides on beef liver glutamate dehydrogenase Effects were observed when experiments were performed in 0.01

M Tris-acetate buffer, pH 8, 10 PM EDTA, and at 25’. Concen- trations of a-ketoglutarate and NH&l were 5 mM and 50 mM,

respectively.

Coenzyme COIlCell- tiation

~~

PM

TPNH 100

DPNH 30

DPNH 100

DPNH 300

-

--

-

GTP or GDP ADP

Inhibition*

Inhibition

Inhibition*

Inhibition

Nuclwtide

Activation*

(34-fold) Activation*

(1.5-fold) Activation

(a-fold) Activation*

(34-fold)

ATP

No effect*

Activation* (1.5-2-fold)

Inhibition

Inhibition*

*Enzymes from other sources were tested, when possible, under these conditions.

Yeast Glutamate Dehydrogenase (TPN-dependent Enzyme)-The enzyme, a gift of Dr. S. Grisolia, was prepared according to the method of Grisolia et al. (8) from fresh bakers’ yeast. The enzyme was kept frozen at -20” and was stable for at least a year under these conditions.

A summary of the purity or of the extent of purification of the enzymes from the different sources is given in Table I.

RESULTS

For simplification, the enzymes from the various sources may be divided into two major classes: those which are influenced by purine nucleotides and those whose activity is unaffected by these compounds.

As with all the glutamate dehydrogenases, assays were performed in 0.01 M Tris-acetate buffer, pH 8. Under these conditions neither the DPN- or TPN- dependent glutamate dehydrogenases from the Neurospora were affected by the purine nucleotides. However, it has been shown that at higher pH values, the DPN-dependent enzyme is inhibited by some of the purine nucleotides. In particular GMP has a (competitive) inhibition constant of approximately 50 PM (11). The difference in results obtained here from those obtained by Sanwal and Lata (9) appears to be mainly due to a difference in pH since the latter experiments were performed at pH 8.5 to 9. These differences are discussed in more detail under “Discussion.”

In any case, it is quite clear that enzymes from nonanimal sources are remarkably different from the enzymes from animal sources as described below. It should be pointed out, and will be discussed in more detail later, that all the enzymes from nonanimal sources are specific for only one of the two coenzymes, DPN or TPN.

Enzymes Afected by Purine Nucleotides

A summary of the effects (activation or inhibition) of various purine nucleotides on the beef liver glutamate dehydrogenase is given in Table II. The effects for which each enzyme (except where noted) were tested are shown by asterisks. Thus, all of the enzymes from animal sources (except that from beef heart) were tested with both coenzymes, TPNH and DPNH, at several levels of DPNH, and with at least three different purine nucleotides. All assays were performed at pH 8, in 0.01 M Tris-acetate buffer (10 PM EDTA) as described above.

From Mammalian Tissues-It has been shown that the level of the glutamate dehydrogenase differs widely in various mammalian tissues (see, for example, Reference 12). Observations in the literature generally agree with those in the present experiments, that is, that liver, kidney, and brain contain considerable amounts of the enzyme while heart has about 10% of the amount, and the amount of enzyme in skeletal muscle is extremely low. All these enzymes catalyze both DPNH and TPNH oxidation, but in some of the crude preparations, notably the beef heart one, there was a considerable amount of DPNH oxidase. All enzymes were purified sufficiently so that essentially no oxidase activity was left, except for the beef heart enzyme, where such activity remained even after some purification. In this case, there was no apparent TPNH oxidase activity and therefore only TPNH was used as the coenzyme when testing for purine nucleotide effects.

In spite of their different levels in the liver, brain, kidney, heart, and skeletal muscle, all the enzymes were affected essentially identically by purine nucleotides. For example, under identical assay conditions, with TPNH as coenzyme, apparent dissociation constants for GTP ranged from 0.2 to 0.5 pM and


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for ADP from 10 to 20 PM for the enzymes from the five sources (Lines 1 and 4 to 7 in Table I).

From Frog and Chicken Liver-& noted in Table I, both the frog and chicken liver glutamate dehydrogenases are crystalline and homogeneous. Preliminary testing of these enzymes showed immediately that they are not identical to one another and that, in addition, they both differ from the beef liver enzyme. De- tailed experiments as to the ratio of velocities in the presence and absence of purine nucleotides and the purine nucleotide binding constants are given in Tables III to VI. In all cases, the results are compared to those previously obtained for the beef liver enzyme and, as indicated above, also obtained for other mammalian enzymes. Tables III and IV represent experiments performed with TPNH as coenzyme. In Table III are shown the ratios of the initial velocities in the presence of purine nucleotide to the velocity in its absence. As can be seen the most marked difference with respect to this ratio is the extent of activation by ADP. Under the conditions used, ADP stimulates both the beef and chicken enzyme, but not the frog liver enzyme. In contrast, the tadpole enzyme appears quit#e similar to the frog liver enzyme.

Table IV gives the apparent binding constants obtained for various purine nucleotides in the presence of 100 pM TPNH. The method for determining these binding constants kinetically has been previously described and consists essentially of deter- mination of the concentration of nucleotide at which the initial velocity is one-half the sum of the velocities in the absence and presence of saturating amounts of nucleotide (2). For the frog liver enzyme, this experiment cannot be performed for ADP since the rat.io of such velocities is 1. However, the binding constant for this nucleotide is easily obtained from competition experiments between ADP and GTP. In this regard it should be pointed out that there is strict competition among these purine nucleotides with all the enzymes tested as had been demonstrated previously for the beef liver enzyme (2).

Table V presents ratios of initial velocities with DPNH as the coenzyme for adenosine di- and triphosphate. Several levels of DPNH have been tested since, as shown in this table and previously, the effects differ depending on the level of the coenzyme (2, 6). For the beef liver enzyme, kinetic evidence for two DPNH binding sites, one active and the other, with less aflinity, nonactive, is obtained from the unusual inhibition of the enzyme by guanosine di- and triphosphate (13) and from the different effects of ATP or DPNH oxidation at different levels of DPNH (2). Table V confirms the previous observation that there is activation at low DPNH levels and inhibition at the high levels of DPNH for the beef liver enzyme. However, while ATP does stimulate the beef liver enzyme activity at very low DPNH concentrations, Table V shows that all the other enzymes tested were inhibited by ATP at these DPNH levels. On the other hand, the extent of inhibition by ATP is greater at the higher DPNH levels.

Table VI lists binding constants obtained for ADP, ATP, and GTP for the beef, frog, and chicken liver enzymes at 100 PM DPNH. Comparison with the binding constants with TPNH as coenzyme (Table IV) shows a number of differences, of which perhaps the largest is the GTP binding constant for the frog liver enzyme.

Table VII lists binding constants for ADP and GDP at three levels of DPNH with the frog, chicken, and beef liver enzymes. For ADP, the binding constants increase from 6- to lo-fold over

May 1965 C. Fr&xkn 2031

a lo-fold increase in the DPNH concentration and are relatively similar to one another. On the other hand, binding constants for GDP either are relatively unchanged (frog) or decrease as the DPNH concentration is increased over the lo-fold range.

TABLE III Ratio of initial velocities in presence and absence of

purine nucleotides (v’/v)

Experiments were performed with TPNH, 100 PM, as coenzyme. Velocities in presence of purine nucleotides were performed at saturating concentration of those nucleotides. Other experimental details were as described in Table II.

VI/V Source

ADP ATP GTP

Beef liver and other mammalian tissues 3.5 1 0.05

Chicken liver 10 l-2 0.05 Frog liver 1 1 40.05 Tadpole liver 1 1 GO.05 Trout liver 2 1 co.1 E. coli Yeast

I

No effects Neurospora

I I

TABLE IV Apparent dissociation constants of purine nucleotides

Experiments were performed at a TPNH concentration of 100 PM. Dissociation constants were calculated as previously described (2). Other experimental conditions are given in Table II.

Dissociation constants Enzyme source

ADP ATP GDP GTP

Beef liver. . . 15 ; l::*pi ;

5 0.4 Chicken liver. . . . 40 10 0.5 Frog liver. . 3* 45 2.0

* Determined by competition experiments. See text.

TABLE V Ratio of initial velocities in presence and absence of

purine nucleotides (v’/v) Experiments were performed with DPNH as coenzyme at satu-

rating concentration of purine nucleotide. Other conditions are described in Table II.

v’/v

SOUFX 30 p?d 100 PY 300 *la

ADP ATP ADP ATP ADP Al-P -- ----

Beef liver and other mam- 1.5-2 1 2 0.6 3.5 0.25 malian tissues

Chicken liver 3 0.6 5 0.3 5 0.2 Frog liver 3.5 0.5 10 0.15 12 0.1 Tadpole liver 0.2 6 0.1 0.12 Trout liver 6 0.7 6 0.5 4.5 0.15 Corn leaf Neurospora (DPN-de- No effects

pendent) II II


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2032 Glutamate Dehydrogenase. VI Vol. 240, No. 5

TABLE VI Apparent dissociation constants for purine nucleotides

Experiments were performed at a DPNH concentration of 100 PM. Dissociation constants, in micromolar, were calculated as previously described (2). Other experimental conditions were as in Table II.

Dissociation constant

ADP ATP GTP

Beef liver. 30 Chicken liver. 60 Frog liver 35 I -

PM

25 0.1 10 0.02 5 0.01

TABLE VII

Effect of DPNH concentration on appa _ .

cr, ent dissociation constants of purine nucleotides for several enzymes

Experiments were performed as described in Table II. Dis- sociation constants, in micromoles per liter, were calculated as previously described (2).

30 PM DPNH 100 PM DPNH 300 ,LP DPNH

ADP GDP ADP GDP ADP ~-

Beef liver 10 5 30 2 100 Chicken liver.. 20 2 60 0.6 130 Frog liver.. 15 0.5 35 0.5 135

_.

-

GDP

2 0.3 0.5

In this case, the binding constants are quite different for the different enzymes as is also the case for GTP.

From Pigeon Liver-The glutamate dehydrogenase from pigeon liver, only about 10% pure (Table I), appeared to be identical with the enzyme obtained from chicken liver. Thus, all the purine nucleotide binding constants and extents of activation or inhibition obtained for the chicken liver enzyme (noted in Tables III to VI) were also obtained for the pigeon liver enzyme.

From Tadpole Liver-Since this enzyme was quite crude, it was not extensively tested. In the preliminary experiments, the enzyme appeared to be quite similar to that obtained from the frog liver, except for the fact that there was considerable inhibition by DPNH at levels higher than 100 PM DPNH. It should be noted that the amount of enzyme in tadpole liver is approximately lo-fold less than in frog liver (per g of tissue). Recently, it has been observed that there are differences in the coenzyme binding constants between the frog and tadpole liver enzymes. These experiments were performed on enzyme which has been more extensively purified than that used in our preliminary experiments.’

From Trout Liver-Glutamate dehydrogenase from trout liver proved to be more difficult to purify than enzyme from other animal livers. Furthermore, in contrast to other impure enzymes, this particular enzyme did not always give consistent results with respect to nucleotide binding constants, DPNH to TPNH activity ratios or the extent of activation or inhibition by the various nucleotides. The results are therefore not as certain as those with the other enzymes. It is not clear why this happens, and it is difficult to state definitely whether this

1 Dr. P. P. Cohen, personal communication.

enzyme is identical to or distinct from other glutamate dehydrogenases.

Effect of EDTA

As previously noted, all experiments reported thus far have been performed in the presence of 10 pM EDTA. However, it has been observed that EDTA has a strong activating effect on the chicken and frog enzyme, although not so marked on the beef liver enzyme. Thus, at 100 pM DPNH, EDTA may activate the frog or chicken liver enzyme 5- to lo-fold but the beef liver enzyme by less than 50%. It seems unlikely that EDTA is removing an inhibitory metal since similar effects are observed even when all solutions are passed over Chelex-100 which removed all the metal ions. Interestingly, the binding constant for the EDTA is extremely low being less than 1 PM. Even more striking is the fact that in the absence of EDTA, ADP will activate the frog and chicken liver enzymes to a much greater extent, but it appears to be bound less tightly. When the enzyme is saturated with EDTA (10 PM), activation by ADP still occurs, as evidenced by Tables III and V. On the other hand, when saturated with ADP (500 ,UM) in the absence of EDTA, no additional activation will occur on addition of EDTA. Although the EDTA effect is not clearly understood, one could postulate a site which binds EDTA fairly specifically. Such a site may also bind ilDP as indicated by the above experiments.

Effect of Zinc

Yielding, Tomkins, and Trundle have shown that the glutamate dehydrogenase from beef liver is strongly inhibited by zinc and that zinc, in the presence of coenzyme will cause (a presumably reversible) dissociation of the enzyme (14). Under the conditions used in the present study, the inhibition constant for zinc is lower than 1 PM for the beef liver enzyme BS well as for the chicken or frog liver enzyme. Other divalent metal ions are not nearly so inhibitory. Binding constants for zinc with the cruder enzyme were obtained, but the values can’only be considered as approximations since there may be other materials which would complex the zinc and make it unavailable to the glutamate dehydrogenase. It is of interest to note that none of t,he enzymes from nonanimal sources were inhibited by these very low concentrations of zinc ions. For this latter class of enzymes, zinc did not inhibit to any appreciable extent at levels less than 200 MM. However, the DPN-dependent enzyme from Neurospora did show a somewhat greater effect. An apparent inhibition constant of about 20 to 30 PM was obtained for this enzyme, although inhibition was not complete even at 200 pM.

All of these experiments were performed, of course, in the absence of EDTA.

DISCUSSION

The primary purpose of the present paper has been to show the similarities and differences which exist with respect to purine nucleotide effects in the glutamate dehydrogenases from various sources. It is evident that there are groups of enzymes which are essentially identical and others which are strikingly different. Thus, the enzymes from mammalian tissues all appear to be the same, but this group is different from those obtained from other animal tissues. The chicken and pigeon liver glutamate dehydrogenases appear to be identical to one another, but different from other classes. The frog liver enzyme again appears to be distinct from other glutamate dehydro-


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May 1965 C. Frieden

genases. By far, the most striking differences are between the enzymes from bacterial or similar sources compared to those from animal tissues, since in t,he former classes there are no distinct purine nucleotide effects.

A few comments are appropriate at this point regarding the differences in the physical properties in some of these enzymes. It has been known for some time that the beef liver glutamate dehydrogenase undergoes a concentration-dependent association- dissociation reaction such that there is a tetramer (possibly a trimer) of active enzyme subunits at high enzyme concentrations (4, 15). Relationships between the degree of association and enzymatic activity have been thoroughly explored and it is fairly well established that various purine nucleotides in the presence of coenzyme may influence the configuration of the active enzyme (2). Although there is some disagreement concerning the molecular weight of the active enzyme, it appears, under our conditions to be about 350,000.2

The chicken liver glutamate dehydrogenase has been shown to have a molecular weight of approximately 430,000 and has been observed to associate only very poorly as the enzyme concentration is raised (17, 18). There is, therefore, a distinct difference between the chicken and beef liver glutamate dehydrogenases with respect to the association-dissociation reaction and it should be pointed out that the data presented in Tables IV and VI show binding constants which are different for the same purine nucleotides, although the differences are not greater than about Bfold. This is also true of the extents of the effect of purine nucleotides on the rate of the reaction starting with either DPNH or TPNH (Tables III and V).

Recently, it has been shown that the crystalline frog liver glutamate dehydrogenase undergoes a concentration-dependent association-dissociation reaction, but that it is a monomer-dimer association, rather than monomer-tetramer (19). The molecular weight of the monomer has been observed to be about 250,000 (19). The effect of purine nucleotides on the reaction rate and the differences in the binding constants of the frog and beef liver enzymes are more striking than the differences between the chicken and beef liver enzyme. Particularly noticeable are the observations that, under the conditions used, ADP did not activate TPNH oxidation (although it is tightly bound) and that the GTP-binding constant is considerably lower for the frog enzyme than for the beef liver enzyme.

It seems appropriate here also to point out that the observed differences in purine nucleotide effects, either in binding constants or extent of effect on activity, may reflect differences in the binding constants of the coenzymes (or substrates) as well as in the purine nucleotide. For example, Fahien, Wiggert and Cohen have shown, and we have also observed, that TPNH is much less tightly bound to the frog liver enzyme than to the beef liver enzyme (19). Thus at 100 PM TPNH, the concentration normally used, the frog liver enzyme is probably not saturated with respect to coenzyme. Similarly, differences might arise from different pH effects, that is one pH curve might be

2 Light scattering data, similar to chat previously publghed (5), have been plotted as ~“-1 against (M,/M, - l)/(n - M,/IM,)“, where c is concentration of protein (milligrams per ml), M, is the weight average molecular weight, Mm is the monomer molecular weight, and n, the number of monomeric units in the polymer (16). A computer was programmed to calculate the terms above for various values of n and M,. The best linear plots were obtained for n = 4 and M,,, = 350,000 i 25,000 (C. Frieden and M. M. Burger, unpublished).

displaced with respect to another (but the shape might be the same). In any case, the essential conclusion is not altered, that the enzymes from the different sources are different in some way.

In this connection, it is interesting to examine the binding constants observed for the various purine nucleotides at the three different DPNH concentrations. From previous kinetic data, one may predict the effect of changing TPNH levels on the binding constants of purine nucleotides and it is found that such binding constants change only 2- to a-fold over a range of 30 to 300 pM TPNH. The changes in binding constants of purine nucleotides when changing DPNH over this same range are much larger. For example, KADp changes about lo-fold for several of the enzymes for this IO-fold increase in DPNH concentration. These larger changes may be attributed to a nonactive DPNH binding site in these enzymes. The fact that not all the animal enzymes behave exactly similarly with respect to different levels of DPNH may indicate the importance or nonimportance of this second site in influencing the kinetic properties of the particular enzyme.

It is clear that the most strikingly different enzymes are those from yeast, bacteria, fungi, and plants. These enzymes, all specific for only one of the two coenzymes are essentially unaffected by purine nucleotides under the present conditions. The observations made so far lead to the generalization that those glutamate dehydrogenases which are specific for one of the two coenzymes do not contain a site which is specific for purine nucleotides as do those glutamate dehydrogenases which can utilize either coenzyme. The DPN-dependent enzyme from Neurospcwa crossa, however, deserves special mention in this regard. It has been shown that this enzyme may be inhibited by a number of purine nucleotides including AMP, GMP, and GTP. In all instances, the inhibition is of a competitive type against either DPN of DPNH (11). Ss yet there appears to be no evidence contrary to the idea that these nucleotides exert their effect by competing with the coenzyme rather than binding to a separate site which is specific for purine nucleotides. In- hibition by GMP, for example, is remarkably strong provided the experiments are performed at about pH 9. We have also observed that AMP at this pH is a much weaker inhibitor. Interestingly enough, we have observed that DPN itself is a very poor inhibitor of the DPNH oxidation compared to the guanosine analogue of DPN which is a very potent inhibitor. These observations indicate a rather unusual specificity of the coenzyme binding site. At lower pH values, for example, pH 8 in 0.01 M Tris-acetate buffer, almost no inhibition can be observed even at concentrations as high as 1 mM. The signifi- cance of this unusual type of pH sensitive inhibition for this enzyme is not clear. Stachow and Sanwal have also pointed out that the TPN dependent enzyme from Neurospora is not affected under the same conditions by these purine nucleotides (11). This observation is reminiscent of the observations by Neufeld, Kaplan, and Colowick (20) that TPN-dependent dehydrogenases are inhibited by 2’-nucleotides, while DPN dependent ones are more specifically inhibited by the 5’-nucleotides. Aside from coenzyme specificity, observed long ago, the glutamate dehydrogenases from such sources appear to be totally different enzymes as attested to by a variety of observations. First, Grisolia et al. (8) have observed the TPN-specific enzyme from yeast to be apparently free of sulfhydryl groups. In contrast, the beef and chicken liver enzymes contain a large number of


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Glutamate Dehydrogenase. VI Vol. 240, No. 5

such groups although they are not detectable in the native enzyme under many conditions. In this connection also, the yeast enzyme appears to be much more stable than the beef liver enzyme. Secondly, the molecular weight of the Neurospma enzyme, for example, is about 265,000 and apparently shows no tendency to associate (21). Third, it has been observed that all the animal enzymes are strongly inhibited by zinc at exceedingly low concentrations. In constrast, low concentrations of zinc have no effect on the enzymes from nonanimal sources.

A single point of similarity between the enzymes from all sources concerns substrate specificity. All the enzymes deami- nate alanine although at a much slower rate than glutamate. Other amino acids as well as their corresponding keto acids which are substrates for the beef liver glutamate dehydrogenases are also substrates for the enzyme from other sources. No detailed study of the comparison of relative rates of a number of substrates for the different enzymes has yet been performed.

It could be argued that the method or degree of purification might influence the kinetic properties of the enzyme from different sources. Reference to their method of purification and to Table I which shows the degree of purification is considerably different for the enzymes from various sources. However, we have observed that the extent of purification does not appear to change the kinetic properties appreciably. For example, com- parisons may be made between partially purified beef, chicken, or frog liver enzyme relative to the crystalline enzymes and no significant differences appear. It is important, however, to test crude preparations for DPNH or TPNH oxidase activity. Such activity was found in very crude preparations, most marked, for example, in the beef heart preparations, and would have obscured the results if undetected since, of course, this activity is unaffected by purine nucleotides. In all of the data presented in this paper, oxidase activities were negligible.

In order to explore whether different purification procedures might affect the results, the beef liver enzyme was prepared by two different procedures. No kinetic difference could be observed when comparing these two preparations. Similarly the chicken and pigeon liver enzymes which differ considerab1.y in the purification procedure as well as in extent of purification (the pigeon is only 10% pure) are almost identical in kinetic parameters.

Another aspect of the present work was an attempt to relate the kinetic properties of the enzymes from different sources to certain aspects of over-all metabolism. For example, it was believed that some differences might exist between the frog liver and the tadpole liver enzyme. Our preliminary results indicated that large differences did not exist. However, recently, Cohen has found some differences between these two enzymes relating to the binding of the coenzyme (either DPN or DPNH) as well as different temperature dependencies of the initial velocity with DPN as the coenzyme.1 Such a difference would be extremely interesting from the point of view of ammonia and urea metabolism, since the frog is ureotelic and the tadpole excretes only ammonia. It was this difference in the two species which initially prompted the investigation of the enzyme from these sources. Also, differences in these enzymes might relate to the observed effects of urea and ammonium on the coenzyme-specific enzymes produced in nonanimal sources. A more general hypothesis regarding this point is discussed below.

The most striking observation to be drawn from the present

results is that those enzymes which are specific for either DPNH of TPNH are not affected specifically by purine nucleotides. In relation to over-all metabolism, there is a different way to state this observation. Thus, one might conclude that in the more complex animal metabolism, the purine nucleotide effects on the glutamate dehydrogenase reaction a$ect the utilization of one of the coenzymes with respect to the other. This hypothesis is supported by the fact that, as shown in this and previous papers, a particular nucleotide may affect the reaction differently depending upon which coenzyme is being used. The most striking example would probably be the frog liver enzyme inhibition by GTP. Comparison of Tables IV and VI shows a 200-fold difference in the inhibition constants for GTP depending on whether DPNH or TPNH is the coenzyme used. Other cases are not nearly so marked although differences in the binding constants of between 2- and lo-fold almost always exist. In such cases where the binding constants for a particular nucleotide to a particular enzyme are not too different, there are differences in the extent of the effect. Thus, ATP has practically no effect on any of the enzymes tested with 100 PM TPNH as coenzyme, but inhibits the reaction from 40 to 90% at the same concentration of DPNH.

Previous results have also shown that DPN and TPN activities are affected differently by the same purine nucleotides, although the differences are not so great (6). Although significant differences between nucleotides in affecting activity or in enzyme binding might be expected to be found in wivo, it is only to be expected that effects of purine nucleotides may be somewhat different than the effects observed under the conditions used in this present work. For this reason, it is difficult to make any detailed correlation between the different kinetic properties and the over-all metabolism of the tissue or organism involved. One other point which should be considered in this regard is the obvious difference in enzyme levels in different tissues. As pointed out previously, for example, skeletal muscle has very little glutamate dehydrogenase compared to the beef liver or kidney.

Finally, in relation to the function of the purine nucleotides in metabolic control, the observations for the Neurospora and the yeast enzyme should be recalled. In the former case, urea represses the level of the TPN-dependent enzyme while inducing formation of the DPN-dependent enzyme (22). In the latter case, ammonia appears to repress the DPN-dependent enzyme without affecting the level of the TPN enzyme (23). The observations may again argue in favor of the hypothesis that the different enzyme activities of the mammalian enzymes may be for different purposes (i.e. different direction of the reaction) and that this differential effect results from the effect of the purine nucleotides on glutamate dehydrogenase.

SUMMARY

1. Glutamate dehydrogenases from a number of sources have been isolated and compared to the beef liver enzyme under particular assay conditions. The assay measures reduced di- or triphosphopyridine nucleotide oxidation in 0.01 M tris(hydroxy- methyl)aminomethaneacetate buffer, pH 8, at 25” in the presence of 10 PM ethylenediaminetetraacetate, 5 mM a-ketogluta-

mate, and 50 mM ammonium chloride. 2. Purine nucleotides strongly and specifically affect the

enzymes from all animal sources examined. However, the binding constants and extent of activation or inhibition differed


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May 1965 C. Frieden 2035

with different enzymes. In general, all the mammalian enzymes appear to be similar with respect to these parameters, whereas the enzyme from other liver sources (chicken, pigeon, frog, tadpole, and trout) were different. In particular, the three crystalline enzymes from beef liver, chicken liver, and frog liver appear to be quite distinct.

3. The glutamate dehydrogenases from nonanimal sources (microorganisms, plant leaves) were in general not influenced by purine nucleotides.

4. Zinc was observed to strongly inhibit the enzyme from all animal sources tested and to have essentially no effect at low concentrations ( < 50 PM) on all the nonanimal enzymes tested.

5. In general, it appears that enzymes specific for only one of the two coenzymes are lacking the specific purine nucleotide and zinc binding sites.

6. A tentative hypothesis that the role of purine nucleotides is to control the rate of utilization of one coenzyme for the reaction (either DPNH or TPNH) relative to the other is proposed.

Acknowledgment-I would like to thank Mr. Frank Falley for excellent technical assistance in performing these experiments.

A&nd~m-Recently Talal and Tomkins (24) have performed immunoelectrophoretic and double diffusion experiments with glutamate dehydrogenases from various sources against antibody made from the beef liver glutamate dehydrogenase. They observed that all of the vertebrate enzymes tested responded to modifiers such as ADP although the bacterial enzymes did not. Furthermore, the bacterial enzymes appeared to be immuno- logically unrelated to the vertebrate enzymes. These experiments are in accord with the experiments reported in this paper. Talal and Ton&ins also noted differences in double diffusion experiments with respect to pigeon liver and rat liver glutamate dehydrogenases. We have shown the differences in the enzyme from pigeon (or chicken) liver compared to the beef liver enzyme above, but differences in the rat liver enzyme were not examined. We have now performed kinetic experiments on this enzyme which, based on the specific activity of the beef liver enzyme, was about 10 to 15% pure. Some differences in the effect of purine nucleotides have been observed, of which the most striking is that of ATP. In contrast to results with enzyme from other mammalian tissues (see Tables III and IV for conditions), ATP

activates TPNH oxidation in rat liver enzyme about g-fold with a binding constant of about 200 MM. In addition, ADP activates TPNH oxidation by the rat liver enzyme about lo-fold instead of 3- to 4-fold for other mammalian enzymes. The binding constant is slightly larger. These and other somewhat smaller differences indicate that the rat liver enzyme is different from other mammalian tissue glutamate dehydrogenases as well as from the enzyme of any other source tested.

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Carl FriedenOTHER EFFECTS ON THE ENZYME FROM VARIOUS SOURCES

Glutamate Dehydrogenase: VI. SURVEY OF PURINE NUCLEOTIDE AND

1965, 240:2028-2035.J. Biol. Chem.

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