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A kinetic study of glutamate dehydrogenase from Xenopus laevis
Int. J. l&hem.,
1974,
Vol. 5, pp. 795 to 805. Pergamon Press. P&ted
A KINETIC
in Great Britain
795
STUDY OF GLUTAMATE DEHYDROGENASE FROM XEJVOPUS LAEVIS ANINA R. LEE
AND
J. B. BALINSRY
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa (Received4
JuneI974)
ABSTRACT I. The ammonia-excreting South African clawed toad Xenopus lamis has a higher activity of glutamate dehydrogenase in the kidney than have other frogs. 2. The kinetics and mechanism of action of glutamate dehydrogenase from the kidney of Xen~pus 1aevi.shave been investigated. The effects of varying concentrations of NAD and glutamate on the velocity of glutamate oxidation and of varying the concentration of NADH, a-ketoglutarate and ammonium on the rate of glutamate synthesis were studied. Product inhibition studies in the direction of glutamate oxidation were performed. In addition, the rate of isotope exchange between [r~C~labelled glutamate and a-ketogiutarate was studied. The results are consistent with an ordered mechanism, with the probable order of addition of substrates in the direction of cu-ketoglutarate reduction being NADH, a-ketoglutarate and finally ammonium ion. 3. Compared to the bovine enzyme, Xeaoptu glutamate dehydrogenase has a high Michaclis constant for ammonium, and a low one for glutamate. This suggests that the function of glutamate dehydrogenase in Xenopw kidney is the formation of excretory ammonia.
EC
beiana (Fahien, Wiggert & Cohen, rg65a), the lungfish (Janssens & Cohen, x968), and the
I ,4. I .3), catalyses the reversible oxidative deamination of glutamate to ol-ketoglutarate and ammonium according to the equation :
dogfish (Corman, Prescott & Kaplan, 1967). It has been purified from a number of micro, organisms, including Thiobacillus novellus (Le
GLUTAMATE dehydrogenase NALt(P)
oxidoreductase
Glutamate
(L-glutamate
(deaminating),
+ NAD(P)+
+ Ha0
:
+ cw-ketoglutarate + NAD(P)H +NH,+ + H+ .
This enzyme also plays a key role in the deamination of many other amino acids (Braunstein & Kritzmann, 1937). It is well known that most amino acids undergo transamination with a-ketoglutarate as the acceptor, thus forming glutamate (Cammarata & Cohen, 1950). Glutamate is then deaminated by glutamate dehydrogenase to produce cu-ketoglutarate and free ammonia. In the reverse process, which is favoured thermodynamically by the reductive amination of a-ketoglutarate, ammonia may be incorporated into glutamate, from which other amino acids can be formed by transamination. The enzyme has been purified from bovine liver (Olson & Anfinsen, x953) and from the liver of the rat (King & Frieden, r970), chicken (Snoke, 1956), the frog Rana cates-
I
John,
Susuki & Wright,
1968) and BlastoKlassen
c~ad~e~~a e~e~sani~ (Le John, Jackson,
St Sawula, r 969). Fahien et al. (1965 a, b) have studied the catalytic properties of glutamate dehydrogenase from the liver of the frog Rana catesbeiana and found them to be similar in many respects to those previously reported for the bovine liver enzyme (Frieden, 1959). The reaction mechanism was found to be identical to that of the beef enzyme. Although the values of the kinetic constants differ slightly from those of bovine liver glutamate dehydrogenase, they are not considered to indicate any difference in the physiological function of the enzyme (Fahien et al., 1965 a).
The South African clawed toad, Xenopus &e&s, is aquatic and excretes predominantly
Int. J. &&hem.
LEE AND BALINSKY
796
ammonia as the end product of amino acid Its kidneys carry out the deacatabolism. mination of many amino acids (Balinsky & Baldwin, 1962). It seems likely that glutamate dehydrogenase functions in the production of excretory ammonia in Xenopus kidneys. The kinetic properties of glutamate dehydrogenase from this source were therefore investigated with this metabolic role in mind. A preliminary report of this work has already been published (Lee & Balinsky, ‘972). MATERIALS
AND
METHODS
REAGENTS NAD, NADH, NADPH, ADP, ATP, L-glutamic acid and a-ketoglutaric acid (monopot~sium salt) were purchased from the Sigma -Chemical Comnanv. St. Louis. MO., U.S.A. Tris iAnalar was bbt&ed from’ the ‘BDH Chemica‘ls Ltd.‘, Poole, U.K. E. Merck Company, Darmstadt, W. Germany, supplied ammonium sulphate and EDTA. whiie PPO and DM-POPOP were obtained -from the Packard Instrument Company, 111.. U.S.A. Glutamic acid. uniformly labelled wi& I%, was obtained from’ The Radcochemical Centre, Amersham, U.K. EXPERIMENTALANIMALS Xenopus laevis were obtained from the Department of Inland Fisheries, Stellenbosch, South Africa. Thev were maintained and fed as described previously (Cragg, Balinsky & Baldwin, 1961). ENZYME ASSAY The assay of glutamate dehydrogenasc activity was carried out using a Gilford zooo recording spectrophotometer and 3 ml. quartz cells with a I cm. light path. Oxidation of NADH was followed by observing the decrease in absorbance at 340 nm. The following assay system was used:- 14 mM o(ketoglutarate, 0.2 mM NADH, 100 pM ADP and 50 mM NH&l contained in a final volume of 3 ml. of 0.1 M Tris-acetate buffer, pH 8.0. The temDerature was qo*C. The reaction was started by be addition oi’ suitably-diluted enzyme. This assay was used routinelv during Durification. For the keverse reaction thk follow
One unit of activity is defined as the amount of enzvme which catalvzes the oroduction or oxidaI I 1~~
tion of I pmole of NADH per minute at 30%. Specific activity is defined as the number of units per mg. of protein, Protein concentration was measured by the method of Warburg & Christian (1942). RELATIVE DISTRIBUTIONOF GLUTAMATE DEKYDROGENASEIN VARIOUS FROGS Enzyme extracts were prepared from liver and kidnev of ~~~~us Laeuis. Bufo redark. Rana andensis a&l R. ~~~~~~1~. ‘I!he Tissu& weie cut LID”and suspended in 20 volumes of cold distilled water and homogenized for I minute using an Ultra-Turrax homogenizer. Mitochondria were ruptured bv ultrasonication. The homogenate was &en centrlfuned for I hour at ~0.000 e in a M.S.E. hieh sp:ed centrifuge at 0’6. TYhe supernatant w>s diluted IO-fold with Tris-acetate buffer, pH 8.0 (0. I M with respect to Tris, containing I x IO--*&~ EDTA) to give a final extract concentration of I in 200 (w/v). The enzyme activities of the various preparations were determined as described above. PARTIAL PURIFICA-I-ION01; GLUTAMATE DEHYDROGENASE FKOM Xenopus laevis The purification procedure was an adaptation of the method used by Corman et al. ( I 967) for the preparation of dogfish glutamate dehydrogenase. Using an Ultra-Turrax homogenizer, Xenopus kidneys were homogenized for 1 minute in IO volumes of cold deionized water. The mitochondria were then further disintegrated by ultrasonication for 5 minutes in a M.S.E. Ultrasonic sonicator operating in the cold room at 4°C. The particulate fractions were removed by ultracentrifugation at 150,ooog in a Spin& Model L ultracentrifuge for I hour. The sedimented material was &shed by resuspending the pellet in distilled water and centrifuging as before. The sunernatant fractions were filtered through glass wOoI to remove fat particles. The clear supernatant fractions from the preceding centrifugation steps were combined and placed on ice. Solid ammonium sulphate was then carefully added, with stirring, to a concentration of q.0 M. The resulting mixture was stirred overniggt at room ternperatlure using a magnetic stirrer. and the resultant Drecioitate collected bv centrifugation at 4,000 g for 20 minutes. Thi’s precipitate was then dissolved in a small volume of 0, I M Tris-acetate buffer. DH 7.6. Undissolved. denatured material was remb;ed by centrifugation: Saturated ammonium sulphate solution, pH 7.0, was added, with stirring, to the concentrated protein solution until it became faintly milky in colour. The solution was, at this stage, about 0.5 M with respect to ammonium sulphate. The enzyme solution was then filtered through glass wool and stored at 4°C for 12 hours for precipitation to proceed. The precipitate was collected by centrifugation and redissolved in the minimum volume of 0.1 M Tris-acetate buffer, pH 7.6. I
1
GLUTAMATE
1974, 5
DEHYDROGENASE
FROM XenO~~S &&S
797
first experiment, the NAD concentration was kept
The enzyme was precipitated three times by the method described above. The enzyme preparation obtained from the repeated precipitation with saturated ammonium sulphate was dialyzed against 0.1 M Tris-acetate buffer, pH 8.0 to remove the ammonium sulphate. To the dialyzed solution was added an equal volume of glycerol with the result that the enzyme was now dissolved in a soIution of 50% (v/v) glycerol-o-o5 M Tris-acetate, pH 8.0. The solution was stored in the deep freeze. The glycerol prevented the solution from freezing. Aliquots of the enzyme preparation were withdrawn and suitably diluted (usually I : 1000) for kinetic studies. A preparation from Xenopus liver was purified by the same method, and was used to repeat some of the experiments in order to check for any differences between the two preparations. XSOTOPICEXCHANGE
The aim of this experiment was to observe the rate of incorporation of 14C into or-ketoglutarate starting with uniformly [14C]labelled glutamate. As this exchange is normally carried out at chemical equilibrium (Cleland, 1967) it was necessary to check the equilibrium concentrations of reactants. This was done by including in the reaction mixture all substrates and products, and varying the concentration of cu-ketoglutarate until no reaction in either direction was observed. The rate of isotope exchange was determined at various concentrations of NH&l, the concentrations of NADH and NAD being suitably adjusted in each case to preserve equilibrium conditions. A stock reaction mixture was prepared containing the reactants at concentrations such that the final reaction mixture would contain 500 PM a-ketogiutarate, ~oopM ADP and r5 mM glutamate containing 0.3 #Ci l*C in O-I M Trisacetate buffer, pH 8.0. For each determination of the exchange rate, an aliquot of the stock mixture was withdrawn. To this was added NH,Cl, NADH and NAD to equilibrium concentrations. The NH,Cl concentration was varied between 5 and zoo mM. In the
constant at IO mM, and the NADH concentration was varied between 2.5 and IOO PM. In the second experiment, NADH was kept at IOO PM, while NH&l and NAD were varied between 5 and 50 mM and IO and IOOPM respectively. At higher concentrations of NH&l, between I oo and ZOO mM, NAD was kept at xoo PM and NADH was varied between 25 and 50 FM. The reaction was started in each case by the addition of enzyme and the reactions were allowed to proceed at 20°C. At different intervals of time, aliquots of the reaction mixture were pipetted directly into 0.1 ml. of 2,4_dinitrophenylhydrazine (5 mg./ml.) in 6 N HCl in order to stop the reaction and to form the 2,+dinitrophenylhydrazone derivative of or-ketoglutarate. The mixtures were allowed to stand at room temperature for 30 minutes. The dinitrophenylhydrazone was then extracted from the aqueous solution by addition of 0.5 ml. ethyl acetate followed by vigorous agitation on a vortex stirrer. The upper ethyl acetate layer was then withdrawn by means of a Pasteur pipette and run into a scintillation vial. This extraction procedure was repeated twice. The combined ethyl acetate fractions were allowed to evaporate to dryness at room temperature in the dark, and the residue dissolved in IO ml. of scintillation fluid (3 g. PPO and 300 mg. of DM-POPOP per litre of toluene). The samples were assayed in a Packard Tri-Garb liquid scintillation counter. The rate of radioactivity incorporation into ar-ketoglutarate is expressed as the number of counts per minute incorporated into a-ketoglutarate in IO minutes.
RESULTS RELATIVE
DISTRIBUTION OF GLUTAMATE
DEHYDROGENASE
ACTIVITY
IN KIDNEY
The relative distribution of glutamate dehydrogenase between the kidney and liver of Xenopus, Rana and Bufo were compared. Table I shows that in Xenopus, the ratio of activity in kidney compared to liver is high;
Table I.-GLUTAMATE
DEHYDROGENASEACTIVITY IN THE LIVER AND KIDNEY OF DIFFERENT SPECIESOF AMPHIHA GLUTAMATE DEHYDROGENASE ACTNI~
(units/g. wet wt., means of 2-6 animals)
k&T10
SPECIES
xetwpus laevis Rana angolem~s Ra~a~u~c~~~a 3ufo regularis
LIVER
850 2700 2580 1620
AND
LIVER
KIDNEY 1380 800 660 ‘35
KIDNEY/LIVER 1.62 O-30 o-26 0.084
LEE AND BALINSKY
798 Table
Int. J. Biochem.
II.-PARTIAL PURIFICATION OF GLUTAMATE DEHYDROGENASE FROM THE KIDNEY OF Xenopus laevis SPECIFIC ACTIVITY (p moles NAD produced per min. per mg. protein)
FRACTION Extract Solid (NH,),SO, precipitate Sat. (NH,),SO, precipitate (I)
0.09
100
0’54
72
2’02
38
22.5
z:
49’0 94
PARTIAL PURIFICATION OF THE ENZYME Table II shows the results of the purification procedure from Xenopus kidney. The extraction was designed to effect considerable purification over the crude homogenate. This purification, however, is not reflected in Table I since protein concentrations of the crude homogenate were not measured. SUBSTRATES
The effect on the velocity of the reaction
;::
varying the concentrations of the components of the reaction mixture was investigated using the partially-purified enzyme preparation from Xenopus kidney. FIG. ~a shows double reciprocal plots (Lineweaver & Burk, 1934) of velocity against NAD concentration at high concentration of glutamate, and the effects of ADP and ATP. The non-linear relationship in the absence of activator was partially abolished by ATP and completely abolished by ADP. FIG. Ib shows the effects of ADP and ATP on the velocity of NADH oxidation when the concentrations of ammonium and a-keto-
in the Rana species, it is much lower, and in Bufb it is very low indeed.
EFFECT OF VARYING
(%)
4’45 8.46
I51
PURIFICATION FACTOR ( - fold) -_____
RECOVERY
of
A
m-
-:::-“ 0
,
.
.
.
iO-
1
/ JO
25
(I
[NAD] -I
(R+.V’
o-
50
100
‘SO
2w
[NADH]-’ W.l)“ b
FIG. I .-The effects of adenine nucleotides on the double reciprocal plots of velocity against coenzyme concentration. Assays were carried out at 30°C in 0.1 M Tris-acetate buffer, pH 8.0, containing IOO FM (a) Reduction of NAD, at 25 mM concentration of glutamate: curve A, in the absence of ATP EDTA. or ADP; curve B, in the presence of IO PM ATP; curve C, in the presence of 5 PM ADP. (b) Oxidation of NADH, the concentrations of a-ketoglutarate and NH,Cl being 14 mM and 50 mM respectively: curve A, in the absence of ADP; curve B, in the presence of IOO PM ADP.
1 Y
FIG. r.--Double reciprocal plots of initial velocity of NAD reduction against concentration of NAD at various fixed levels of glutamate, The concentrations of glutamate were: A, 2 mM; B, 5 mM; C, IO mM; D, zo mM; E, extrapolated to infinite concentration. Assays were carried out at 30°C in 0.1 M Tris-acetate buffer, pH 8.0, containing IOOuhl EDTA and roe ,uM ADP.
glutarate were kept high. At high concentrations of NADH, substrate inhibition occurred. ADP is an activator of the reaction and also partially counteracts the substrate inhibition by NADH. All subsequent experiments were carried out in the presence of roopM ADP,
The double recipraca1 plots of velocity against NAD concentration at various constant levels of glutamate, shown in FIG. t2, give an intersecting pattern. FIG. 3a represents a plot of reciprocal velocity against reciprocal NADH concentration at several fixed fevels of cu-ketoghztarate. Ammonium ions were kept at a constant saturating level. A family of lines intersecting on the abscissa was obtained. When the concentrations of NADH and ammonium were varied at saturating iw-ketoglutarate levels, the double reciprocal plots wrere parallel (Fro. 3b). At high IeveIs of NADH, the pIot of reciprocaY: velocity against reciprocal or-ketoglutarate concentration at several different concentrations of ammonium is intersecting, though the point of intersection is far to the left of the ordinate (FIG. 3~). The FIG. g.--Double reciprocal plots of initial veiocity of NADH oxidation against substrate concentration. (a) Variation of NADH concentration at constant NH&l concentration (50 mM) and
rY
cc52 1 ”
/
&
1.0
[dl&
I.0
(mM)J
at several fixed concentrations of a-ketoglutarate :
A, 0.7 mM; B, 1.4 mM; C, 2.8 mM; D, 4’7 mM;
E, 7 mM; F, extrapolated to infinite concentration. (b) Variation of NADH concentration at constant concentration of a-ketoglutarate ($4 mM) and at several fixed concentrations of NH&I: A, 8.35 mM; B, 12.5 mM; C, qj mM; D, jjo mM; E, extrapolated to infinite concentration. (c) Variation of a-ketoglutarate concentration at constant NADH concentration (zoo PM) and at several fixed concentrations of NH&l: A, 5 mM; B, 8.33 mM; C, 12.5 nM; D, 25 mM; E, extrapolated to infinite concentration. (d) Variatian of oc-ketoglutarate concentration in the high range, at constant concentrations of NADH (zoo PM) and NH&I (50 mM) _ Assays were carried out at go”C in o-f M T&acetate buffer, PH 8.0, containing IOO FM EDTA and 100 FM ADP.
inset shows that rY-ketoglutarate, like NADIR, is an inhibitor of the enzyme at high concentrations. This inhibition is probably due to the formation of the ‘dead-end’ complex
E-NAD-or-ketoglutarate (Cross, McGregor & Fisher, 1g62). When a preparation of the enzyme from X8nopu.f liver was used, the patterns obtained were similar to those shown in FIGS. 3a, b, and c.
The inhibition of the reaction in the direction of NAD reduction was studied in order to provide further information about the mechanism of the reaction (Cleland, 1963 b, c). NAD was the variable substrate, and the concentration of glutamate was kept constant. FIG. 4a shows the inhibition by NADH to be competitive for NAD, while that of ~-ketoglutarate is rmcompetitive (FIG. 4b). The significance of the anomalous fine at high ol-ketoglutarate concentration will be discussed below. Ammonium was a non-competitive inhibitor with respect to NAD (FIG. 4~). Again, similar patterns were obtained when a preparation from .Ye3zi$usliver was used. The data of the experiments on the effect ofsubstrate concentration and those on product inhibition were used to calculate respectively Michaelis and inhibition constants according to the appropriate computer programme of Cleland (1963 d). These values are shown in Table III. No marked differences between the values for kidney and liver were observed. hX’OPE
EXCHANGE
The isotope exchange between gIutamate and a-ketoglutarate was measured under chemical equilibrium conditions. The value of the equilibrium constant
F’rc. +-Inhibition of NAD reduction by products of the reaction, Glutamate concentration was IOO mM. The remaining experimental conditions were as described for FIG. 2. (a) Inhibition by NADH at concentrations of: A, IOO PM; B, 66 p&l; C, 40 PM; D, zero. (b) Inhibition by &etoglutarate, at concentrations of: A, 14 mM; B, 9.7 mM; C, 7 mM; D, 3.5 mM; E, zero. (c) ~*bi~~tion by NH&l, at concentrations of: A, P?C; mM: B. 17 mM: C. IO mM: D, zero.
was found to be ro-x4. Published values for this constant vary between I x IO-~~ and I x 10-l~ (Frieden, 1959~). In the exchange experiments, the concentration of NH,CI was varied between 5 and zoo $vI and NAD or NADH concentration was adjusted to keep ~uil~briunl conditions. It had been ascertained, prior to conducting this experiment,
GLUTAMATE
DEHYDROGENASE
FROM &nofaUS
801
hVis
1
100
%lSI
I I I I I
I
m
[NH,']-*M-I Frc.5_---Double reciprocal plot of the rate of isotope exchange between [‘4C]glutamate ancl tiketoglutarate against NH &I concentration, using kidney GIXI. The experimental conditions are described in Methods. that the high concentrations of NH&l did not inhibit the enzyme. Similar results were obtained in both exchange experiments; Fro. 5 shows the results of the second experiment. Between 5 and 50 mM NH,Cl, the rate uf exchange increased with NH&l concentration; at ~~nc~~trat~ons higher thau 50 mM, inhibition of the exchange rate was observed. DISCUSSION Glutamate dehydrogenase occupies a central position in metabolism. It connects ammonia, the primary product of amino acid deamination and a substrate for many enzymes, with glutamate, which is the common metabolite in a large number of transamination reactions. The enzyme could thus play a role in a variety of metabolic processes, and many theories about its function have been postulated. Thus Braunstein & Kritzmann (I 93 7) formulated a theory for deamination of amino acids in which a-ketoglutarate acts as an acceptor of a-amino groups by
802
LEE AND BALINSKY
transamination, and glutamate dehydrogenase converts the resulting glutamate to ammonia. On the other hand Cohen (1966) summarized the evidence that in ureotelic animals, glutamate dehydrogenase is an accessory enzyme to the urea cycle, capturing free ammonia which is subsequently converted into the amino group of aspartate. This is supported by the fact that the activity of glutamate dehydrogenase increases in tadpoles of Rana catesbeiana at metamorphosis, together with the urea cycle enzymes (De Groot & Cohen, 1962) and is induced by thyroxine treatment in uiuo and in vitro (Balinsky et al., ‘970). Different theories of the metabolic role of glutamate dehydrogenase hinge on whether the enzyme works in the direction of glutamate oxidation, of glutamate synthesis, or in either direction. The equilibrium constant of the reaction favours glutamate synthesis. This may not, however, be a decisive factor in any given system, as the living cell is in a steady state rather than in equilibrium, and continual removal of metabolites may promote a reaction not favoured by the equilibrium. Other evidence must be considered in order to decide the predominant metabolic One important consideration is the route. location of the enzyme in the organism. Thus kidney glutamate dehydrogenase is unlikely to participate in urea synthesis, as the Krebs urea cycle is absent from that organ. On the other hand, the enzyme is favourably Iocated there for the formation of excretory ammonia. If glutamate dehydrogenase has a special function in producing ammonia in Xen@us, then it is of interest to compare the distribution of glutamate dehydrogenase in Xenopus with that in Rana, which excretes mainly urea and some ammonia, and in Bufo which excretes even less ammonia than IZana (Cragg et al., x96x; Balinsky, 1970). The results in Table I suggest a correlation between the presence of glutamate dehydrogenase in the kidneys and the amount of ammonia excreted. By contrast, the activity of glutaminase in the kidney of Xen0pu.s does not appear to be higher than in Rana (Balinsky & Baldwin, 1962). This is in accord with the
Int.
J.
Biochem.
view that ghrtamine is not as important a precursor of ammonia in Amphibia as it is in mammals (Balinsky, I 970). A further important consideration are the values for the Michaelis constants for ammonium and glutamate. Compared to glutamate dehydrogenase of other species, that of Xenopus laevis appears to have a low K;, for glutamate (400 l_tM) and a high K, for ammonium (20,000 PM) (Table III). The possibility exists that the values of these constants are altered in the intra-mitochondrial conditions, and by the concentrations of activating and inhibiting nucleotides in viuo. Nevertheless, it seems likely that a high concentration of ammonium is needed for glutamate to be formed, while oxidation of glutamate can occur at relatively low concentrations of this substance. In the kidney cells, the concentration of ammonium is likely to be low as a result of the excretion of this compound by the kidney. At low concentrations of reactants in the cell, a high Michaelis constant for ammonium would favour the production of ammonium, rather than its utilization. The function of the relatively large amount of glutamate dehydrogenase in Xenopus kidney compared to the liver is thus probably the formation of ammonia by the excretory organ. The activation of the reaction in both directions by ADP and ATP (FIG. I) is similar to that found for glutamate dehydrogenase from the liver of the American bullfrog Rana catesbeiancs (Fahien et al., rg65 b). The activating effect ofADP on Xenopus glutamate dehydrogenase, and the elimination of nonlinearity of the double reciprocal plots with NAD and glutamate as substrates was used in the present work in order to simplify the kinetic behaviour of the enzymes. Frieden (1959) proposed a sequential ordered mechanism for ghrtamate dehydrogenase, and the evidence of several other workers (Fahien et al., 1965 a; Corman et al., 1967 ; Le John et al., 1968, 1968) is in agreement with this assumption. Engel & Dalziel (I 969) and Fisher and collaborators (Cross et al., 1972; Colen, Prough & Fisher, 1972) on the other hand, have postulated a random mechanism. The present results as
GLUTAMATE DEHYDROGENASE FROM &lO~US
‘9749 5
discussed below are consistent with an ordered mechanism operating under the assay conditions employed by us. When the concentrations of NAD and glutamate were varied (FIG. 2) a pattern of intersecting lines was obtained which indicate that the mechanism in the direction of NAD reduction is sequential, i.e. both substrates must be present before any product is released. If it is assumed that the coenzyme reacts with the free enzyme (as has been proposed for bovine and Rana enzyme), and that an ordered or rapid equilibrium random mechanism operates, then the point of intersection of the lines in this figure gives the value of the dissociation constant of the enzyme-NAD complex. Since the lines intersect above the abscissa, the dissociation constant is higher than the Michaelis constant for NAD (Table III). Because the reaction in the direction of glutamate synthesis involves three substrates, it is possible to obtain some evidence about the mechanism from kinetic data (Frieden I g5g a, b, c) . The intersection of the family of lines in FIG. 3a on the abscissa resembles the results of Frieden (I g5g a, b, c) for bovine glutamate dehydrogenase and of Fahien et al. ( I 965 a) for the enzyme from Rana catesbeiana. If NADH is the first substrate to be added, this result means that the Michaelis constant for NADH is the same as the dissociation constant of the E-NADH complex. FIGS. 3b and c, however, show a different result for Xenopus glutamate dehydrogenase compared to1 the enzyme from other verteNAD(P)
Glu
heViS
803
brates. Varying NADH and ammonium resulted in a parallel pattern of lines with Xenopus enzyme (FIG. gb), while that from ox (Frieden, 1959) Rana (Fahien et al., 1965 a, b) and dogfish (Corman et al., 1967) gave an intersecting pattern. When NADH and 01ketoglutarate were varied (FIG. 3c), an intersecting family of lines resulted with Xenopus glutamate dehydrogenase, while other vertebrates gave parallel lines. The patterns in Xenopus are, in fact similar to those obtained for the NAD-specific glutamate dehydrogenase from Thiobacillus novellus (Le John et al., 1968). The results suggest that, in Xenopus glutamate dehydrogenase, c+ketoglutarate is added between NADH and NH,+. If NADH is the first substrate to be added in this direction, it follows that NH,+ is the last, or in the other direction, the first, product to be released. Using the notation of Cleland (1963 a), the mechanism proposed by Frieden can be represented by Scheme I. The enzyme from Rana catesbeiana (Fahien Ig65a, b) and from the dogfish et al., (Corman et al., 1967) gave results consistent with this mechanism. In the NAD-specific glutamate dehydrogenase from Thiobacillus novellus (Le John et al., 1g68), from Blastocladiella emersonii (Le John et al., rg6g) and from JVeurospora crassa (Stachow, rg65), the results point to ammonium, and not a-ketoglutarate, as the first product released. This mechanism can be represented by Scheme 2.
aKg
NH,+
t
t
NAD(P)H
t
I I _~.._J ~. _.~_ mm_mmm_.~_4 _.~ E
E-NAD (P)
E-NAD(P)-Glu E-NAD(P)H! k arKg-NH,+
E-NAD(P)H‘! NH,+ j
E
E-NAD(P)H
SCHEME I NAD
NH,+
Glu
NADH
a%
t I 4 E
E-NAG-
T E-NAD-Glu
I E-NADH[ dg-NH,+
E-NADH4? SCHEME 2
_~ A E-NADH
_
_~ ~~~_~~ ~~_ E
go4
LEE AND BALINSKY
The results of the present paper are consistent with the second rather than the first mechanism. The same order of addition is suggested by the patterns of product inhibition (FIG. 4). NADH is competitive with NAD (FIG. da), probably combining with free enzyme. w Ketoglutarate is an uncompetitive inhibitor, presumably reacting with E-NADH. The parallel pattern (FIG. 4b) results from the fact that, under the conditions of the experiment, the release of ammonium is irreversible, and the enzyme forms reacting with NAD and a-ketoglutarate are not connected by reversible reactions (Cleland, 1963~). The altered slope of the line obtained at the highest concentration of ~-ketoglutarate is probably due to the formation of the ‘dead end’ E-NAD-or-ketoglutarate complex (Cross et al., 1972). Ammonium is a non-competitive inhibitor versus NAD (FIG. 4c), probably combining with E-NADH-a-ketoglutarate. A ‘dead-end’ E-NAD-NH,+ complex cannot be ruled out, though there is no evidence of substrate inhibition by ammonium ions. The proposed reaction sequence for Xenopus glutamate dehydrogenase is strengthened by the study of isotope exchange between glutamate and cu-ketoglutarate in the presence of the enzyme. The rate of incorporation of radioactivity into a-ketoglutarate at chemical equilibrium was measured as a function of ammonia concentration (FIG. 5). The inhibition of the exchange at high concentrations of NH,+ indicates that the release of this product by the enzyme is an event which occurs after the binding of glutamate, and before the release of a-ketoglutarate. Saturation with ammonium, therefore, inhibits the reversible reaction between glutamate and ol-ketoglutarate. All the evidence discussed above is tonsistent with an ordered reaction mechanism. It is less easy to reconciie with a random mechanism, which has also been postulated (Engel & Dalziel, 1969; Colen et al., 1972) for the bovine enzyme. It should be pointed out that our experiments were carried out in the presence of the activator ADP, which could result in a different reaction mechanAlso, the less favourable substrate ism.
KADP
ht. J. Biochem.
rather
than NAD was used by Colen The above interpretation must, therefore, be restricted to the conditions used in the present experiments. The difference of the present results from those of Frieden ( I 959) cannot be ascribed to any differences in the experimental conditions, for when the experiments shown in FIG. 3 were repeated on bovine liver glutamate dehydrogenase they gave results similar to those of Frieden (1959) (Lee & Balinsky, unpublished results). The difference is also not due to the fact that NADH rather than NADPH was used in the present experiments, since NADPH, although a very poor substrate for Xenopus glutamate dehydrogenase, gave results similar to NADH (Lee & Balinsky, unpublished results). Nor is the proposed mechanism for the Xe?lopus enzyme due to an organ-specific difference, since liver and kidney enzymes showed identical patterns and similar constants (Table III). If there is a difference in the mechanism of Xenopus glutamate dehydrogenase, it is tempting to speculate that the difference has some significance in an animal which produces large amounts of ammonia. If glutamate dehydrogenase plays an important role in the formation of ammonia in Xenopus, then the special properties of the enzyme may be regarded as adaptations to ammonia production.
et al. (I 972).
This work was supported in part by grants from the Council for Scientific and Industrial Research and the Atomic Energy Board. REFERENCES BALINSKY, J. B. (Igyo), ‘ Nitrogen metabolism in Amphibians ‘, in Comparative Biochemisty oj ~itr~~e~ metabolism (Ed. Campbell), Vol. z, pp. 5rg-637. London & New York: Academic Press. BALINSKY, J. B., & BALDWIN,E. (rg62), ‘ Comparative studies of waste nitrogen metabolism in Amphibia ‘, Biochem. J., 82, 187-191. BALINSKY, J. B., SIIAMBAUGH, G. E., & COHEN, B. B. (I970), ‘ Glutamate dehydrogenase biosynthesis in Amphibian liver preparation ‘, J. Biol. Chem., 245, 128-137. BRAUNSTEIN, A. E., & KRITZMANN,M. G. (Ig37), ‘ Formation and breakdown of amino acids by intermolecular transfer of the amino group ‘, JWure, Lo&., ‘40, 503-504.
‘9742 5
GLUTAMATE
DEHYDROGENASE
P. S., & COHEN, P. P. (Ig5o), ‘ The scope of the transamination reaction in animal tissues ‘, 3. Biol. Chem., 187, 439-452. CLELAND, W. W. (Ig63a), ‘ The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations ‘, Biochim. Biophys. Acta, 67, 104-137. ‘ The kinetics of CLELAND, W. W. (Ig63b), enzyme-cai.alyzed reactions with two or more substrates or products. II. Inhibition: nomenclature ancl theory ‘, Biochim. Biophys. Acta, 67, 173-187. ‘ The kinetics of CLELAND, W. W. (Ig63c), enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection ‘, Biochim. Biophys. Acta, 67, 188-196. W. (Ig63d), ‘ Computer proCLELAND, W. grammes for processing enzyme kinetic data’, Nature, Lond., 198, 463-465. CLELAND, W. W. (Ig67), ‘ Enzyme Kinetics ‘, A. Rev. Biochem., 36, 77-I I 2. ‘ Biochemical aspects of COHEN, P. I’. (Ig66), metamorphosis ‘, Harvey Lect., 60, 119-154. COLEN, A. H., PROUGH, R. A., & FISCHER, H. F. ‘ The mechanism of glutamate de(I972)> hydrogenase reaction. IV. Evidence for random and rapid binding of substrate and coenzyme in the burst phase ‘, 3, Biol. Chem., 247, 7go5-7909. CORMAN, L. Sr KAPLAN, N. 0. (Ig67), ‘ Kinetic studies of dogfish liver glutamate dehydrogenase with diphosphopyridine nucleotide and the effect of added salts ‘, 3. Biol. Chem., 242, 28402846. CORMAN, L., PRESCOTT, L. M., & KAPLAN, N. 0. (rg67), ‘ Purification and kinetic characteristics of dogfish liver glutamate dehydrogenase ‘, 3. Biol. Chem., 242, 1383-1390. CRAGG, M. h/Z., BALINSKY, J. B., & BALDWIN, E. (1g61), ‘ A comparative study of nitrogen excretion in some Amphibia and Reptiles ‘. Camp. Biochem. Physiol., 3, 227-235. CROSS, D. G., MCGREGOR, L. T., & FISHER, H. F. (rg72), ‘ The binding of ar-ketoglutarate in a binary complex with NADP+ by L-glutamate dehydrogen ase ‘, Biochim. Biophys. Acta, 289, 28-36. DE GROOT, N., & COHEN, P. P. (rg6z), ‘ Studies on dehydrogenase activities during amphibian metamorphosis ‘, Biochim. Biophys. Acta, 59,
CAMMARATA,
588-594. ENGEL, P. C., & DALZIEL, K. (Ig6g), ‘ The kinetic studies of glutamate dehydrogenase. The reductive amination of n-oxoglutarate ‘, Biothem. J., 1x8, 409-419.
FROM
&XOpUS
hZViS
805
FAAIEN, L. A., WIGGERT, B. O., & COHEN, P. P. (Ig65a), ‘ Crystallization and kinetic properties of glutamate dehydrogenase from frog liver ‘, 3. Biol. Chem., 240, 1083-1090. FAHIEN, L. A., WIGGERT, B. O., & COHEN, P. P. (Ig65b), ‘ Effect of nucleotides and coenzymes on frog liver glutamate dehydrogenase ‘, 3. Biol. Chem., 240, IogI-Iog5. FRIEDEN, C. (1959 a), ‘Glutamic dehydrogenase. I. The effect of coenzyme on the sedimentation velocity and kinetic behaviour ‘, 3. Biol. Chem., 234,8og-8 I 4. FRIEDEN, C. (1959 b), ‘ Glutamic dehydrogenase. II. The effect of various nucleotides on the association-dissociation and kinetic properties ‘, 3. Biol. Chem., 234,815-820. ‘ Glutamic dehydrogenase. FRIEDEN, C. (Igsgc), III. The order of substrate addition in the enzymatic reaction ‘, 3. Biol. Chem., 234, 28gI-
2896. JANSSENS,P. A., & COHEN, P. P. (Ig68), ‘ Nitrogen metabolism in the African lunefish ‘. Comb. Biochem. Physiol., 24, 879-886. v ‘ I KING, K. S., & FRIEDEN, C. (1g7o), ‘ The purification and physical properties of glutamate dehydrogenase from rat liver ‘, 3. Biol. Chem., 245, 4391-4396. LE TOHN. H. B.. SUSUKI, I.. & WRIGHT. 1. A. ‘ Glutamate dkhyhrogenases ok -7hiobacillus novellus ‘, 3. Biol. Chem., 243, I I8-128. LE JOHN, H. B., JACKSON, S. G., KLASSEN, G. R., & SAWULA, R. V. (Ig6g), ‘ Regulation of mitochondrial glutamic dehydrogenase by divalent metals, nucleotides and a-ketoglutarate ‘, 3. Biol. Chem., 244, 5346-5356. LEE, A. R., & BALINSKY, J. B. (1972). ‘ The kinetic properties of glutamatedehy drogenase from Xenopus laevis ‘, S. Afr. Med. J., 46, 779. LINEWEAVER, H., & BURK, D. (rg34), ‘ The determination of enzyme dissociation constants ‘, 3. Am. Chem. Sot., 56, 658-666. OLSON, J. A., & ANFINSEN, C. B. (tg53), ‘ The crystallization and characterization of glutamic acid dehydrogenase ‘, 3. Biol. Chem., rg7,67-79. SNOKE,J. E. (Ig56), ‘ Chicken liver glutamate dehydrogenase ‘, 3. Biol. Chem., 223, 27 1-276. STACHOW, C. S. (Ig65), Ph.D. Thesis, University of Manitoba, Quoted by Le John et al., 1969. WARBURG, D., & CHRISTIAN, W. (Ig42), ‘ Isolation und Kristallisation der Enolaae ‘, Biochem. Z’. ,310,384-42 I.
(I-@-U;
Key Word Index: Amphibia, Xenopus laevis, glutamate dehydrogenase, kinetics, isotope cxchange.
1974,
Vol. 5, pp. 795 to 805. Pergamon Press. P&ted
A KINETIC
in Great Britain
795
STUDY OF GLUTAMATE DEHYDROGENASE FROM XEJVOPUS LAEVIS ANINA R. LEE
AND
J. B. BALINSRY
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa (Received4
JuneI974)
ABSTRACT I. The ammonia-excreting South African clawed toad Xenopus lamis has a higher activity of glutamate dehydrogenase in the kidney than have other frogs. 2. The kinetics and mechanism of action of glutamate dehydrogenase from the kidney of Xen~pus 1aevi.shave been investigated. The effects of varying concentrations of NAD and glutamate on the velocity of glutamate oxidation and of varying the concentration of NADH, a-ketoglutarate and ammonium on the rate of glutamate synthesis were studied. Product inhibition studies in the direction of glutamate oxidation were performed. In addition, the rate of isotope exchange between [r~C~labelled glutamate and a-ketogiutarate was studied. The results are consistent with an ordered mechanism, with the probable order of addition of substrates in the direction of cu-ketoglutarate reduction being NADH, a-ketoglutarate and finally ammonium ion. 3. Compared to the bovine enzyme, Xeaoptu glutamate dehydrogenase has a high Michaclis constant for ammonium, and a low one for glutamate. This suggests that the function of glutamate dehydrogenase in Xenopw kidney is the formation of excretory ammonia.
EC
beiana (Fahien, Wiggert & Cohen, rg65a), the lungfish (Janssens & Cohen, x968), and the
I ,4. I .3), catalyses the reversible oxidative deamination of glutamate to ol-ketoglutarate and ammonium according to the equation :
dogfish (Corman, Prescott & Kaplan, 1967). It has been purified from a number of micro, organisms, including Thiobacillus novellus (Le
GLUTAMATE dehydrogenase NALt(P)
oxidoreductase
Glutamate
(L-glutamate
(deaminating),
+ NAD(P)+
+ Ha0
:
+ cw-ketoglutarate + NAD(P)H +NH,+ + H+ .
This enzyme also plays a key role in the deamination of many other amino acids (Braunstein & Kritzmann, 1937). It is well known that most amino acids undergo transamination with a-ketoglutarate as the acceptor, thus forming glutamate (Cammarata & Cohen, 1950). Glutamate is then deaminated by glutamate dehydrogenase to produce cu-ketoglutarate and free ammonia. In the reverse process, which is favoured thermodynamically by the reductive amination of a-ketoglutarate, ammonia may be incorporated into glutamate, from which other amino acids can be formed by transamination. The enzyme has been purified from bovine liver (Olson & Anfinsen, x953) and from the liver of the rat (King & Frieden, r970), chicken (Snoke, 1956), the frog Rana cates-
I
John,
Susuki & Wright,
1968) and BlastoKlassen
c~ad~e~~a e~e~sani~ (Le John, Jackson,
St Sawula, r 969). Fahien et al. (1965 a, b) have studied the catalytic properties of glutamate dehydrogenase from the liver of the frog Rana catesbeiana and found them to be similar in many respects to those previously reported for the bovine liver enzyme (Frieden, 1959). The reaction mechanism was found to be identical to that of the beef enzyme. Although the values of the kinetic constants differ slightly from those of bovine liver glutamate dehydrogenase, they are not considered to indicate any difference in the physiological function of the enzyme (Fahien et al., 1965 a).
The South African clawed toad, Xenopus &e&s, is aquatic and excretes predominantly
Int. J. &&hem.
LEE AND BALINSKY
796
ammonia as the end product of amino acid Its kidneys carry out the deacatabolism. mination of many amino acids (Balinsky & Baldwin, 1962). It seems likely that glutamate dehydrogenase functions in the production of excretory ammonia in Xenopus kidneys. The kinetic properties of glutamate dehydrogenase from this source were therefore investigated with this metabolic role in mind. A preliminary report of this work has already been published (Lee & Balinsky, ‘972). MATERIALS
AND
METHODS
REAGENTS NAD, NADH, NADPH, ADP, ATP, L-glutamic acid and a-ketoglutaric acid (monopot~sium salt) were purchased from the Sigma -Chemical Comnanv. St. Louis. MO., U.S.A. Tris iAnalar was bbt&ed from’ the ‘BDH Chemica‘ls Ltd.‘, Poole, U.K. E. Merck Company, Darmstadt, W. Germany, supplied ammonium sulphate and EDTA. whiie PPO and DM-POPOP were obtained -from the Packard Instrument Company, 111.. U.S.A. Glutamic acid. uniformly labelled wi& I%, was obtained from’ The Radcochemical Centre, Amersham, U.K. EXPERIMENTALANIMALS Xenopus laevis were obtained from the Department of Inland Fisheries, Stellenbosch, South Africa. Thev were maintained and fed as described previously (Cragg, Balinsky & Baldwin, 1961). ENZYME ASSAY The assay of glutamate dehydrogenasc activity was carried out using a Gilford zooo recording spectrophotometer and 3 ml. quartz cells with a I cm. light path. Oxidation of NADH was followed by observing the decrease in absorbance at 340 nm. The following assay system was used:- 14 mM o(ketoglutarate, 0.2 mM NADH, 100 pM ADP and 50 mM NH&l contained in a final volume of 3 ml. of 0.1 M Tris-acetate buffer, pH 8.0. The temDerature was qo*C. The reaction was started by be addition oi’ suitably-diluted enzyme. This assay was used routinelv during Durification. For the keverse reaction thk follow
One unit of activity is defined as the amount of enzvme which catalvzes the oroduction or oxidaI I 1~~
tion of I pmole of NADH per minute at 30%. Specific activity is defined as the number of units per mg. of protein, Protein concentration was measured by the method of Warburg & Christian (1942). RELATIVE DISTRIBUTIONOF GLUTAMATE DEKYDROGENASEIN VARIOUS FROGS Enzyme extracts were prepared from liver and kidnev of ~~~~us Laeuis. Bufo redark. Rana andensis a&l R. ~~~~~~1~. ‘I!he Tissu& weie cut LID”and suspended in 20 volumes of cold distilled water and homogenized for I minute using an Ultra-Turrax homogenizer. Mitochondria were ruptured bv ultrasonication. The homogenate was &en centrlfuned for I hour at ~0.000 e in a M.S.E. hieh sp:ed centrifuge at 0’6. TYhe supernatant w>s diluted IO-fold with Tris-acetate buffer, pH 8.0 (0. I M with respect to Tris, containing I x IO--*&~ EDTA) to give a final extract concentration of I in 200 (w/v). The enzyme activities of the various preparations were determined as described above. PARTIAL PURIFICA-I-ION01; GLUTAMATE DEHYDROGENASE FKOM Xenopus laevis The purification procedure was an adaptation of the method used by Corman et al. ( I 967) for the preparation of dogfish glutamate dehydrogenase. Using an Ultra-Turrax homogenizer, Xenopus kidneys were homogenized for 1 minute in IO volumes of cold deionized water. The mitochondria were then further disintegrated by ultrasonication for 5 minutes in a M.S.E. Ultrasonic sonicator operating in the cold room at 4°C. The particulate fractions were removed by ultracentrifugation at 150,ooog in a Spin& Model L ultracentrifuge for I hour. The sedimented material was &shed by resuspending the pellet in distilled water and centrifuging as before. The sunernatant fractions were filtered through glass wOoI to remove fat particles. The clear supernatant fractions from the preceding centrifugation steps were combined and placed on ice. Solid ammonium sulphate was then carefully added, with stirring, to a concentration of q.0 M. The resulting mixture was stirred overniggt at room ternperatlure using a magnetic stirrer. and the resultant Drecioitate collected bv centrifugation at 4,000 g for 20 minutes. Thi’s precipitate was then dissolved in a small volume of 0, I M Tris-acetate buffer. DH 7.6. Undissolved. denatured material was remb;ed by centrifugation: Saturated ammonium sulphate solution, pH 7.0, was added, with stirring, to the concentrated protein solution until it became faintly milky in colour. The solution was, at this stage, about 0.5 M with respect to ammonium sulphate. The enzyme solution was then filtered through glass wool and stored at 4°C for 12 hours for precipitation to proceed. The precipitate was collected by centrifugation and redissolved in the minimum volume of 0.1 M Tris-acetate buffer, pH 7.6. I
1
GLUTAMATE
1974, 5
DEHYDROGENASE
FROM XenO~~S &&S
797
first experiment, the NAD concentration was kept
The enzyme was precipitated three times by the method described above. The enzyme preparation obtained from the repeated precipitation with saturated ammonium sulphate was dialyzed against 0.1 M Tris-acetate buffer, pH 8.0 to remove the ammonium sulphate. To the dialyzed solution was added an equal volume of glycerol with the result that the enzyme was now dissolved in a soIution of 50% (v/v) glycerol-o-o5 M Tris-acetate, pH 8.0. The solution was stored in the deep freeze. The glycerol prevented the solution from freezing. Aliquots of the enzyme preparation were withdrawn and suitably diluted (usually I : 1000) for kinetic studies. A preparation from Xenopus liver was purified by the same method, and was used to repeat some of the experiments in order to check for any differences between the two preparations. XSOTOPICEXCHANGE
The aim of this experiment was to observe the rate of incorporation of 14C into or-ketoglutarate starting with uniformly [14C]labelled glutamate. As this exchange is normally carried out at chemical equilibrium (Cleland, 1967) it was necessary to check the equilibrium concentrations of reactants. This was done by including in the reaction mixture all substrates and products, and varying the concentration of cu-ketoglutarate until no reaction in either direction was observed. The rate of isotope exchange was determined at various concentrations of NH&l, the concentrations of NADH and NAD being suitably adjusted in each case to preserve equilibrium conditions. A stock reaction mixture was prepared containing the reactants at concentrations such that the final reaction mixture would contain 500 PM a-ketogiutarate, ~oopM ADP and r5 mM glutamate containing 0.3 #Ci l*C in O-I M Trisacetate buffer, pH 8.0. For each determination of the exchange rate, an aliquot of the stock mixture was withdrawn. To this was added NH,Cl, NADH and NAD to equilibrium concentrations. The NH,Cl concentration was varied between 5 and zoo mM. In the
constant at IO mM, and the NADH concentration was varied between 2.5 and IOO PM. In the second experiment, NADH was kept at IOO PM, while NH&l and NAD were varied between 5 and 50 mM and IO and IOOPM respectively. At higher concentrations of NH&l, between I oo and ZOO mM, NAD was kept at xoo PM and NADH was varied between 25 and 50 FM. The reaction was started in each case by the addition of enzyme and the reactions were allowed to proceed at 20°C. At different intervals of time, aliquots of the reaction mixture were pipetted directly into 0.1 ml. of 2,4_dinitrophenylhydrazine (5 mg./ml.) in 6 N HCl in order to stop the reaction and to form the 2,+dinitrophenylhydrazone derivative of or-ketoglutarate. The mixtures were allowed to stand at room temperature for 30 minutes. The dinitrophenylhydrazone was then extracted from the aqueous solution by addition of 0.5 ml. ethyl acetate followed by vigorous agitation on a vortex stirrer. The upper ethyl acetate layer was then withdrawn by means of a Pasteur pipette and run into a scintillation vial. This extraction procedure was repeated twice. The combined ethyl acetate fractions were allowed to evaporate to dryness at room temperature in the dark, and the residue dissolved in IO ml. of scintillation fluid (3 g. PPO and 300 mg. of DM-POPOP per litre of toluene). The samples were assayed in a Packard Tri-Garb liquid scintillation counter. The rate of radioactivity incorporation into ar-ketoglutarate is expressed as the number of counts per minute incorporated into a-ketoglutarate in IO minutes.
RESULTS RELATIVE
DISTRIBUTION OF GLUTAMATE
DEHYDROGENASE
ACTIVITY
IN KIDNEY
The relative distribution of glutamate dehydrogenase between the kidney and liver of Xenopus, Rana and Bufo were compared. Table I shows that in Xenopus, the ratio of activity in kidney compared to liver is high;
Table I.-GLUTAMATE
DEHYDROGENASEACTIVITY IN THE LIVER AND KIDNEY OF DIFFERENT SPECIESOF AMPHIHA GLUTAMATE DEHYDROGENASE ACTNI~
(units/g. wet wt., means of 2-6 animals)
k&T10
SPECIES
xetwpus laevis Rana angolem~s Ra~a~u~c~~~a 3ufo regularis
LIVER
850 2700 2580 1620
AND
LIVER
KIDNEY 1380 800 660 ‘35
KIDNEY/LIVER 1.62 O-30 o-26 0.084
LEE AND BALINSKY
798 Table
Int. J. Biochem.
II.-PARTIAL PURIFICATION OF GLUTAMATE DEHYDROGENASE FROM THE KIDNEY OF Xenopus laevis SPECIFIC ACTIVITY (p moles NAD produced per min. per mg. protein)
FRACTION Extract Solid (NH,),SO, precipitate Sat. (NH,),SO, precipitate (I)
0.09
100
0’54
72
2’02
38
22.5
z:
49’0 94
PARTIAL PURIFICATION OF THE ENZYME Table II shows the results of the purification procedure from Xenopus kidney. The extraction was designed to effect considerable purification over the crude homogenate. This purification, however, is not reflected in Table I since protein concentrations of the crude homogenate were not measured. SUBSTRATES
The effect on the velocity of the reaction
;::
varying the concentrations of the components of the reaction mixture was investigated using the partially-purified enzyme preparation from Xenopus kidney. FIG. ~a shows double reciprocal plots (Lineweaver & Burk, 1934) of velocity against NAD concentration at high concentration of glutamate, and the effects of ADP and ATP. The non-linear relationship in the absence of activator was partially abolished by ATP and completely abolished by ADP. FIG. Ib shows the effects of ADP and ATP on the velocity of NADH oxidation when the concentrations of ammonium and a-keto-
in the Rana species, it is much lower, and in Bufb it is very low indeed.
EFFECT OF VARYING
(%)
4’45 8.46
I51
PURIFICATION FACTOR ( - fold) -_____
RECOVERY
of
A
m-
-:::-“ 0
,
.
.
.
iO-
1
/ JO
25
(I
[NAD] -I
(R+.V’
o-
50
100
‘SO
2w
[NADH]-’ W.l)“ b
FIG. I .-The effects of adenine nucleotides on the double reciprocal plots of velocity against coenzyme concentration. Assays were carried out at 30°C in 0.1 M Tris-acetate buffer, pH 8.0, containing IOO FM (a) Reduction of NAD, at 25 mM concentration of glutamate: curve A, in the absence of ATP EDTA. or ADP; curve B, in the presence of IO PM ATP; curve C, in the presence of 5 PM ADP. (b) Oxidation of NADH, the concentrations of a-ketoglutarate and NH,Cl being 14 mM and 50 mM respectively: curve A, in the absence of ADP; curve B, in the presence of IOO PM ADP.
1 Y
FIG. r.--Double reciprocal plots of initial velocity of NAD reduction against concentration of NAD at various fixed levels of glutamate, The concentrations of glutamate were: A, 2 mM; B, 5 mM; C, IO mM; D, zo mM; E, extrapolated to infinite concentration. Assays were carried out at 30°C in 0.1 M Tris-acetate buffer, pH 8.0, containing IOOuhl EDTA and roe ,uM ADP.
glutarate were kept high. At high concentrations of NADH, substrate inhibition occurred. ADP is an activator of the reaction and also partially counteracts the substrate inhibition by NADH. All subsequent experiments were carried out in the presence of roopM ADP,
The double recipraca1 plots of velocity against NAD concentration at various constant levels of glutamate, shown in FIG. t2, give an intersecting pattern. FIG. 3a represents a plot of reciprocal velocity against reciprocal NADH concentration at several fixed fevels of cu-ketoghztarate. Ammonium ions were kept at a constant saturating level. A family of lines intersecting on the abscissa was obtained. When the concentrations of NADH and ammonium were varied at saturating iw-ketoglutarate levels, the double reciprocal plots wrere parallel (Fro. 3b). At high IeveIs of NADH, the pIot of reciprocaY: velocity against reciprocal or-ketoglutarate concentration at several different concentrations of ammonium is intersecting, though the point of intersection is far to the left of the ordinate (FIG. 3~). The FIG. g.--Double reciprocal plots of initial veiocity of NADH oxidation against substrate concentration. (a) Variation of NADH concentration at constant NH&l concentration (50 mM) and
rY
cc52 1 ”
/
&
1.0
[dl&
I.0
(mM)J
at several fixed concentrations of a-ketoglutarate :
A, 0.7 mM; B, 1.4 mM; C, 2.8 mM; D, 4’7 mM;
E, 7 mM; F, extrapolated to infinite concentration. (b) Variation of NADH concentration at constant concentration of a-ketoglutarate ($4 mM) and at several fixed concentrations of NH&I: A, 8.35 mM; B, 12.5 mM; C, qj mM; D, jjo mM; E, extrapolated to infinite concentration. (c) Variation of a-ketoglutarate concentration at constant NADH concentration (zoo PM) and at several fixed concentrations of NH&l: A, 5 mM; B, 8.33 mM; C, 12.5 nM; D, 25 mM; E, extrapolated to infinite concentration. (d) Variatian of oc-ketoglutarate concentration in the high range, at constant concentrations of NADH (zoo PM) and NH&I (50 mM) _ Assays were carried out at go”C in o-f M T&acetate buffer, PH 8.0, containing IOO FM EDTA and 100 FM ADP.
inset shows that rY-ketoglutarate, like NADIR, is an inhibitor of the enzyme at high concentrations. This inhibition is probably due to the formation of the ‘dead-end’ complex
E-NAD-or-ketoglutarate (Cross, McGregor & Fisher, 1g62). When a preparation of the enzyme from X8nopu.f liver was used, the patterns obtained were similar to those shown in FIGS. 3a, b, and c.
The inhibition of the reaction in the direction of NAD reduction was studied in order to provide further information about the mechanism of the reaction (Cleland, 1963 b, c). NAD was the variable substrate, and the concentration of glutamate was kept constant. FIG. 4a shows the inhibition by NADH to be competitive for NAD, while that of ~-ketoglutarate is rmcompetitive (FIG. 4b). The significance of the anomalous fine at high ol-ketoglutarate concentration will be discussed below. Ammonium was a non-competitive inhibitor with respect to NAD (FIG. 4~). Again, similar patterns were obtained when a preparation from .Ye3zi$usliver was used. The data of the experiments on the effect ofsubstrate concentration and those on product inhibition were used to calculate respectively Michaelis and inhibition constants according to the appropriate computer programme of Cleland (1963 d). These values are shown in Table III. No marked differences between the values for kidney and liver were observed. hX’OPE
EXCHANGE
The isotope exchange between gIutamate and a-ketoglutarate was measured under chemical equilibrium conditions. The value of the equilibrium constant
F’rc. +-Inhibition of NAD reduction by products of the reaction, Glutamate concentration was IOO mM. The remaining experimental conditions were as described for FIG. 2. (a) Inhibition by NADH at concentrations of: A, IOO PM; B, 66 p&l; C, 40 PM; D, zero. (b) Inhibition by &etoglutarate, at concentrations of: A, 14 mM; B, 9.7 mM; C, 7 mM; D, 3.5 mM; E, zero. (c) ~*bi~~tion by NH&l, at concentrations of: A, P?C; mM: B. 17 mM: C. IO mM: D, zero.
was found to be ro-x4. Published values for this constant vary between I x IO-~~ and I x 10-l~ (Frieden, 1959~). In the exchange experiments, the concentration of NH,CI was varied between 5 and zoo $vI and NAD or NADH concentration was adjusted to keep ~uil~briunl conditions. It had been ascertained, prior to conducting this experiment,
GLUTAMATE
DEHYDROGENASE
FROM &nofaUS
801
hVis
1
100
%lSI
I I I I I
I
m
[NH,']-*M-I Frc.5_---Double reciprocal plot of the rate of isotope exchange between [‘4C]glutamate ancl tiketoglutarate against NH &I concentration, using kidney GIXI. The experimental conditions are described in Methods. that the high concentrations of NH&l did not inhibit the enzyme. Similar results were obtained in both exchange experiments; Fro. 5 shows the results of the second experiment. Between 5 and 50 mM NH,Cl, the rate uf exchange increased with NH&l concentration; at ~~nc~~trat~ons higher thau 50 mM, inhibition of the exchange rate was observed. DISCUSSION Glutamate dehydrogenase occupies a central position in metabolism. It connects ammonia, the primary product of amino acid deamination and a substrate for many enzymes, with glutamate, which is the common metabolite in a large number of transamination reactions. The enzyme could thus play a role in a variety of metabolic processes, and many theories about its function have been postulated. Thus Braunstein & Kritzmann (I 93 7) formulated a theory for deamination of amino acids in which a-ketoglutarate acts as an acceptor of a-amino groups by
802
LEE AND BALINSKY
transamination, and glutamate dehydrogenase converts the resulting glutamate to ammonia. On the other hand Cohen (1966) summarized the evidence that in ureotelic animals, glutamate dehydrogenase is an accessory enzyme to the urea cycle, capturing free ammonia which is subsequently converted into the amino group of aspartate. This is supported by the fact that the activity of glutamate dehydrogenase increases in tadpoles of Rana catesbeiana at metamorphosis, together with the urea cycle enzymes (De Groot & Cohen, 1962) and is induced by thyroxine treatment in uiuo and in vitro (Balinsky et al., ‘970). Different theories of the metabolic role of glutamate dehydrogenase hinge on whether the enzyme works in the direction of glutamate oxidation, of glutamate synthesis, or in either direction. The equilibrium constant of the reaction favours glutamate synthesis. This may not, however, be a decisive factor in any given system, as the living cell is in a steady state rather than in equilibrium, and continual removal of metabolites may promote a reaction not favoured by the equilibrium. Other evidence must be considered in order to decide the predominant metabolic One important consideration is the route. location of the enzyme in the organism. Thus kidney glutamate dehydrogenase is unlikely to participate in urea synthesis, as the Krebs urea cycle is absent from that organ. On the other hand, the enzyme is favourably Iocated there for the formation of excretory ammonia. If glutamate dehydrogenase has a special function in producing ammonia in Xen@us, then it is of interest to compare the distribution of glutamate dehydrogenase in Xenopus with that in Rana, which excretes mainly urea and some ammonia, and in Bufo which excretes even less ammonia than IZana (Cragg et al., x96x; Balinsky, 1970). The results in Table I suggest a correlation between the presence of glutamate dehydrogenase in the kidneys and the amount of ammonia excreted. By contrast, the activity of glutaminase in the kidney of Xen0pu.s does not appear to be higher than in Rana (Balinsky & Baldwin, 1962). This is in accord with the
Int.
J.
Biochem.
view that ghrtamine is not as important a precursor of ammonia in Amphibia as it is in mammals (Balinsky, I 970). A further important consideration are the values for the Michaelis constants for ammonium and glutamate. Compared to glutamate dehydrogenase of other species, that of Xenopus laevis appears to have a low K;, for glutamate (400 l_tM) and a high K, for ammonium (20,000 PM) (Table III). The possibility exists that the values of these constants are altered in the intra-mitochondrial conditions, and by the concentrations of activating and inhibiting nucleotides in viuo. Nevertheless, it seems likely that a high concentration of ammonium is needed for glutamate to be formed, while oxidation of glutamate can occur at relatively low concentrations of this substance. In the kidney cells, the concentration of ammonium is likely to be low as a result of the excretion of this compound by the kidney. At low concentrations of reactants in the cell, a high Michaelis constant for ammonium would favour the production of ammonium, rather than its utilization. The function of the relatively large amount of glutamate dehydrogenase in Xenopus kidney compared to the liver is thus probably the formation of ammonia by the excretory organ. The activation of the reaction in both directions by ADP and ATP (FIG. I) is similar to that found for glutamate dehydrogenase from the liver of the American bullfrog Rana catesbeiancs (Fahien et al., rg65 b). The activating effect ofADP on Xenopus glutamate dehydrogenase, and the elimination of nonlinearity of the double reciprocal plots with NAD and glutamate as substrates was used in the present work in order to simplify the kinetic behaviour of the enzymes. Frieden (1959) proposed a sequential ordered mechanism for ghrtamate dehydrogenase, and the evidence of several other workers (Fahien et al., 1965 a; Corman et al., 1967 ; Le John et al., 1968, 1968) is in agreement with this assumption. Engel & Dalziel (I 969) and Fisher and collaborators (Cross et al., 1972; Colen, Prough & Fisher, 1972) on the other hand, have postulated a random mechanism. The present results as
GLUTAMATE DEHYDROGENASE FROM &lO~US
‘9749 5
discussed below are consistent with an ordered mechanism operating under the assay conditions employed by us. When the concentrations of NAD and glutamate were varied (FIG. 2) a pattern of intersecting lines was obtained which indicate that the mechanism in the direction of NAD reduction is sequential, i.e. both substrates must be present before any product is released. If it is assumed that the coenzyme reacts with the free enzyme (as has been proposed for bovine and Rana enzyme), and that an ordered or rapid equilibrium random mechanism operates, then the point of intersection of the lines in this figure gives the value of the dissociation constant of the enzyme-NAD complex. Since the lines intersect above the abscissa, the dissociation constant is higher than the Michaelis constant for NAD (Table III). Because the reaction in the direction of glutamate synthesis involves three substrates, it is possible to obtain some evidence about the mechanism from kinetic data (Frieden I g5g a, b, c) . The intersection of the family of lines in FIG. 3a on the abscissa resembles the results of Frieden (I g5g a, b, c) for bovine glutamate dehydrogenase and of Fahien et al. ( I 965 a) for the enzyme from Rana catesbeiana. If NADH is the first substrate to be added, this result means that the Michaelis constant for NADH is the same as the dissociation constant of the E-NADH complex. FIGS. 3b and c, however, show a different result for Xenopus glutamate dehydrogenase compared to1 the enzyme from other verteNAD(P)
Glu
heViS
803
brates. Varying NADH and ammonium resulted in a parallel pattern of lines with Xenopus enzyme (FIG. gb), while that from ox (Frieden, 1959) Rana (Fahien et al., 1965 a, b) and dogfish (Corman et al., 1967) gave an intersecting pattern. When NADH and 01ketoglutarate were varied (FIG. 3c), an intersecting family of lines resulted with Xenopus glutamate dehydrogenase, while other vertebrates gave parallel lines. The patterns in Xenopus are, in fact similar to those obtained for the NAD-specific glutamate dehydrogenase from Thiobacillus novellus (Le John et al., 1968). The results suggest that, in Xenopus glutamate dehydrogenase, c+ketoglutarate is added between NADH and NH,+. If NADH is the first substrate to be added in this direction, it follows that NH,+ is the last, or in the other direction, the first, product to be released. Using the notation of Cleland (1963 a), the mechanism proposed by Frieden can be represented by Scheme I. The enzyme from Rana catesbeiana (Fahien Ig65a, b) and from the dogfish et al., (Corman et al., 1967) gave results consistent with this mechanism. In the NAD-specific glutamate dehydrogenase from Thiobacillus novellus (Le John et al., 1g68), from Blastocladiella emersonii (Le John et al., rg6g) and from JVeurospora crassa (Stachow, rg65), the results point to ammonium, and not a-ketoglutarate, as the first product released. This mechanism can be represented by Scheme 2.
aKg
NH,+
t
t
NAD(P)H
t
I I _~.._J ~. _.~_ mm_mmm_.~_4 _.~ E
E-NAD (P)
E-NAD(P)-Glu E-NAD(P)H! k arKg-NH,+
E-NAD(P)H‘! NH,+ j
E
E-NAD(P)H
SCHEME I NAD
NH,+
Glu
NADH
a%
t I 4 E
E-NAG-
T E-NAD-Glu
I E-NADH[ dg-NH,+
E-NADH4? SCHEME 2
_~ A E-NADH
_
_~ ~~~_~~ ~~_ E
go4
LEE AND BALINSKY
The results of the present paper are consistent with the second rather than the first mechanism. The same order of addition is suggested by the patterns of product inhibition (FIG. 4). NADH is competitive with NAD (FIG. da), probably combining with free enzyme. w Ketoglutarate is an uncompetitive inhibitor, presumably reacting with E-NADH. The parallel pattern (FIG. 4b) results from the fact that, under the conditions of the experiment, the release of ammonium is irreversible, and the enzyme forms reacting with NAD and a-ketoglutarate are not connected by reversible reactions (Cleland, 1963~). The altered slope of the line obtained at the highest concentration of ~-ketoglutarate is probably due to the formation of the ‘dead end’ E-NAD-or-ketoglutarate complex (Cross et al., 1972). Ammonium is a non-competitive inhibitor versus NAD (FIG. 4c), probably combining with E-NADH-a-ketoglutarate. A ‘dead-end’ E-NAD-NH,+ complex cannot be ruled out, though there is no evidence of substrate inhibition by ammonium ions. The proposed reaction sequence for Xenopus glutamate dehydrogenase is strengthened by the study of isotope exchange between glutamate and cu-ketoglutarate in the presence of the enzyme. The rate of incorporation of radioactivity into a-ketoglutarate at chemical equilibrium was measured as a function of ammonia concentration (FIG. 5). The inhibition of the exchange at high concentrations of NH,+ indicates that the release of this product by the enzyme is an event which occurs after the binding of glutamate, and before the release of a-ketoglutarate. Saturation with ammonium, therefore, inhibits the reversible reaction between glutamate and ol-ketoglutarate. All the evidence discussed above is tonsistent with an ordered reaction mechanism. It is less easy to reconciie with a random mechanism, which has also been postulated (Engel & Dalziel, 1969; Colen et al., 1972) for the bovine enzyme. It should be pointed out that our experiments were carried out in the presence of the activator ADP, which could result in a different reaction mechanAlso, the less favourable substrate ism.
KADP
ht. J. Biochem.
rather
than NAD was used by Colen The above interpretation must, therefore, be restricted to the conditions used in the present experiments. The difference of the present results from those of Frieden ( I 959) cannot be ascribed to any differences in the experimental conditions, for when the experiments shown in FIG. 3 were repeated on bovine liver glutamate dehydrogenase they gave results similar to those of Frieden (1959) (Lee & Balinsky, unpublished results). The difference is also not due to the fact that NADH rather than NADPH was used in the present experiments, since NADPH, although a very poor substrate for Xenopus glutamate dehydrogenase, gave results similar to NADH (Lee & Balinsky, unpublished results). Nor is the proposed mechanism for the Xe?lopus enzyme due to an organ-specific difference, since liver and kidney enzymes showed identical patterns and similar constants (Table III). If there is a difference in the mechanism of Xenopus glutamate dehydrogenase, it is tempting to speculate that the difference has some significance in an animal which produces large amounts of ammonia. If glutamate dehydrogenase plays an important role in the formation of ammonia in Xenopus, then the special properties of the enzyme may be regarded as adaptations to ammonia production.
et al. (I 972).
This work was supported in part by grants from the Council for Scientific and Industrial Research and the Atomic Energy Board. REFERENCES BALINSKY, J. B. (Igyo), ‘ Nitrogen metabolism in Amphibians ‘, in Comparative Biochemisty oj ~itr~~e~ metabolism (Ed. Campbell), Vol. z, pp. 5rg-637. London & New York: Academic Press. BALINSKY, J. B., & BALDWIN,E. (rg62), ‘ Comparative studies of waste nitrogen metabolism in Amphibia ‘, Biochem. J., 82, 187-191. BALINSKY, J. B., SIIAMBAUGH, G. E., & COHEN, B. B. (I970), ‘ Glutamate dehydrogenase biosynthesis in Amphibian liver preparation ‘, J. Biol. Chem., 245, 128-137. BRAUNSTEIN, A. E., & KRITZMANN,M. G. (Ig37), ‘ Formation and breakdown of amino acids by intermolecular transfer of the amino group ‘, JWure, Lo&., ‘40, 503-504.
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GLUTAMATE
DEHYDROGENASE
P. S., & COHEN, P. P. (Ig5o), ‘ The scope of the transamination reaction in animal tissues ‘, 3. Biol. Chem., 187, 439-452. CLELAND, W. W. (Ig63a), ‘ The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations ‘, Biochim. Biophys. Acta, 67, 104-137. ‘ The kinetics of CLELAND, W. W. (Ig63b), enzyme-cai.alyzed reactions with two or more substrates or products. II. Inhibition: nomenclature ancl theory ‘, Biochim. Biophys. Acta, 67, 173-187. ‘ The kinetics of CLELAND, W. W. (Ig63c), enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection ‘, Biochim. Biophys. Acta, 67, 188-196. W. (Ig63d), ‘ Computer proCLELAND, W. grammes for processing enzyme kinetic data’, Nature, Lond., 198, 463-465. CLELAND, W. W. (Ig67), ‘ Enzyme Kinetics ‘, A. Rev. Biochem., 36, 77-I I 2. ‘ Biochemical aspects of COHEN, P. I’. (Ig66), metamorphosis ‘, Harvey Lect., 60, 119-154. COLEN, A. H., PROUGH, R. A., & FISCHER, H. F. ‘ The mechanism of glutamate de(I972)> hydrogenase reaction. IV. Evidence for random and rapid binding of substrate and coenzyme in the burst phase ‘, 3, Biol. Chem., 247, 7go5-7909. CORMAN, L. Sr KAPLAN, N. 0. (Ig67), ‘ Kinetic studies of dogfish liver glutamate dehydrogenase with diphosphopyridine nucleotide and the effect of added salts ‘, 3. Biol. Chem., 242, 28402846. CORMAN, L., PRESCOTT, L. M., & KAPLAN, N. 0. (rg67), ‘ Purification and kinetic characteristics of dogfish liver glutamate dehydrogenase ‘, 3. Biol. Chem., 242, 1383-1390. CRAGG, M. h/Z., BALINSKY, J. B., & BALDWIN, E. (1g61), ‘ A comparative study of nitrogen excretion in some Amphibia and Reptiles ‘. Camp. Biochem. Physiol., 3, 227-235. CROSS, D. G., MCGREGOR, L. T., & FISHER, H. F. (rg72), ‘ The binding of ar-ketoglutarate in a binary complex with NADP+ by L-glutamate dehydrogen ase ‘, Biochim. Biophys. Acta, 289, 28-36. DE GROOT, N., & COHEN, P. P. (rg6z), ‘ Studies on dehydrogenase activities during amphibian metamorphosis ‘, Biochim. Biophys. Acta, 59,
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FAAIEN, L. A., WIGGERT, B. O., & COHEN, P. P. (Ig65a), ‘ Crystallization and kinetic properties of glutamate dehydrogenase from frog liver ‘, 3. Biol. Chem., 240, 1083-1090. FAHIEN, L. A., WIGGERT, B. O., & COHEN, P. P. (Ig65b), ‘ Effect of nucleotides and coenzymes on frog liver glutamate dehydrogenase ‘, 3. Biol. Chem., 240, IogI-Iog5. FRIEDEN, C. (1959 a), ‘Glutamic dehydrogenase. I. The effect of coenzyme on the sedimentation velocity and kinetic behaviour ‘, 3. Biol. Chem., 234,8og-8 I 4. FRIEDEN, C. (1959 b), ‘ Glutamic dehydrogenase. II. The effect of various nucleotides on the association-dissociation and kinetic properties ‘, 3. Biol. Chem., 234,815-820. ‘ Glutamic dehydrogenase. FRIEDEN, C. (Igsgc), III. The order of substrate addition in the enzymatic reaction ‘, 3. Biol. Chem., 234, 28gI-
2896. JANSSENS,P. A., & COHEN, P. P. (Ig68), ‘ Nitrogen metabolism in the African lunefish ‘. Comb. Biochem. Physiol., 24, 879-886. v ‘ I KING, K. S., & FRIEDEN, C. (1g7o), ‘ The purification and physical properties of glutamate dehydrogenase from rat liver ‘, 3. Biol. Chem., 245, 4391-4396. LE TOHN. H. B.. SUSUKI, I.. & WRIGHT. 1. A. ‘ Glutamate dkhyhrogenases ok -7hiobacillus novellus ‘, 3. Biol. Chem., 243, I I8-128. LE JOHN, H. B., JACKSON, S. G., KLASSEN, G. R., & SAWULA, R. V. (Ig6g), ‘ Regulation of mitochondrial glutamic dehydrogenase by divalent metals, nucleotides and a-ketoglutarate ‘, 3. Biol. Chem., 244, 5346-5356. LEE, A. R., & BALINSKY, J. B. (1972). ‘ The kinetic properties of glutamatedehy drogenase from Xenopus laevis ‘, S. Afr. Med. J., 46, 779. LINEWEAVER, H., & BURK, D. (rg34), ‘ The determination of enzyme dissociation constants ‘, 3. Am. Chem. Sot., 56, 658-666. OLSON, J. A., & ANFINSEN, C. B. (tg53), ‘ The crystallization and characterization of glutamic acid dehydrogenase ‘, 3. Biol. Chem., rg7,67-79. SNOKE,J. E. (Ig56), ‘ Chicken liver glutamate dehydrogenase ‘, 3. Biol. Chem., 223, 27 1-276. STACHOW, C. S. (Ig65), Ph.D. Thesis, University of Manitoba, Quoted by Le John et al., 1969. WARBURG, D., & CHRISTIAN, W. (Ig42), ‘ Isolation und Kristallisation der Enolaae ‘, Biochem. Z’. ,310,384-42 I.
(I-@-U;
Key Word Index: Amphibia, Xenopus laevis, glutamate dehydrogenase, kinetics, isotope cxchange.