Kinetic properties of glutamate dehydrogenase purified from the mealworm fat body. The glutamate synthesizing direction

Kinetic properties of glutamate dehydrogenase purified from the mealworm fat body. The glutamate synthesizing direction

Comp. Biochem. PhysioL Vol. 90B, No. 2, pp. 329-333, 1988 Printed in Great Britain 0305-0491/88 $3.00+ 0.00 © 1988PergamonPress plc KINETIC PROPERTI...

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Comp. Biochem. PhysioL Vol. 90B, No. 2, pp. 329-333, 1988 Printed in Great Britain

0305-0491/88 $3.00+ 0.00 © 1988PergamonPress plc

KINETIC PROPERTIES OF GLUTAMATE DEHYDROGENASE PURIFIED FROM THE MEALWORM FAT BODY. THE GLUTAMATE SYNTHESIZING DIRECTION JAN K. TELLER Department of Animal Physiology, A. Mickiewicz University, Fredry 10, 61-701 Poznafi, Poland (Received 20 July 1987)

Abstract--l. Glutamate dehydrogenase (GDH) purified from the mealworm fat body was investigated in the glutamate synthesizing direction. 2. Kinetic constants for the NADH and NADPH linked reactions were determined in the presence and absence of the activator, ADP. 3. ADP has influenced maximal velocities, substrate constants and cooperativity coefficients. 4. Strong substrate inhibition was observed at high concentrations of all substrates. 5. GDH was also activated by leucine and EDTA, and inhibited by zinc ions. 6. The enzyme displays allosteric properties being precisely controlled by substrates and effector concentrations. 7. The kinetic properties of Tenebrio molitor GDH suggest the regulatory role of the enzyme in nitrogen and carbohydrate metabolism of the fat body.

INTRODUCTION Glutamate dehydrogenase (GDH) (EC 1.4.1.3) catalyses the reductive amination of 2-oxoglutarate to glutamate as well as the oxidative deamination of L-glutamate. The enzyme can function in incorporating ammonium ion and the catabolism of glutamate. Thus it occupies the central position in metabolism of amino acids and carbohydrates (Goldin and Frieden, 1971; Smith et al., 1975). In insects the role of G D H is probably manifold and depends on the tissue metabolism and the stage of development. The difference between insect GDHs are mainly manifested in kinetic behaviour because the enzyme resembles structurally other animal GDHs (Caggese et al., 1982; Male and Storey, 1982, 1983; Teller, 1987). Detailed description of kinetic properties of G D H from the fat body is one of the ways to establish the significance of this enzyme in insect metabolism. In the present study GDH purified to molecular homogeneity from the Tenebrio molitor fat body has been characterized kinetically in the glutamate synthesizing direction. MATERIALS AND METHODS

Studies were carried out on 100-130nag larvae of Tenebrio mofitor L. (Insecta : Coleoptera) reared as outlined by Teller and Pile 0985). The purification of GDH from the fat body was performed as described recently (Teller, 1987). Enzyme activity was measured at 25°C using a computercontrolled spectrophotometer following the absorbance at 340 nm. Assay conditions were as follows. NADH linked activity was assayed in 50 mM triethanolamine-HCI-NaOH buffer, pH 7.5, 0-0.5 mM NADH, 0-50 mM 2-oxoglutarate, 0--750raM NH4Cl and 0-2raM ADP. NADPH linked activity was assayed in 50 mM imidazole-HCl buffer, pH 6.75, 0-0.2 mM NADPH, 0-5 mM 2-oxoglutarate, 0-100raM NH4C1 and 0-0.5raM ADP. Reactions were C.B.P. 90/2B---F

329

started by the addition of enzyme at the concentration range of 0.3-2.0/~g/ml. Initial rates were calculated using the molar extinction coefficient for NAD(P)H of 6.2 x 106cm2/mol. Enzyme activity was expressed in katals. The protein content of the enzyme preparation was determined by the method of Bradford (1976) using bovine GDH as a standard. Maximal velocities, I'm, substrate affinities, K0.5,and Hill coefficients,h, were determined by the method of Endrenyi et al. (1975). Reagents were purchased from: bovine liver GDH, NADPH, NADH and sodium 2-oxoglutarate--Sigma, USA; ammonium chloride and imidazole--Merck, FRG. Other chemicals were from POCh, Poland. RESULTS N A D H linked activity

In order to study the kinetic properties of mealworm G D H the effects of each substrate at the saturating concentrations of the others were studied. Figure 1 shows the effect of varied ammonium ion concentration on the velocity of reductive amination reaction in the presence or absence of 1 mM ADP. This concentration of ADP gave the maximal activation in optimal conditions (Fig. 4). In the absence of ADP the sigmoidal shape of the saturation curve was observed. ADP activated the enzyme at all substrate concentrations studied, with the maximal effect of about two-fold activation at 200 mM concentration of ammonium ion. Substrate inhibition was observed at high concentration of the substrate on both curves. Optimal concentration of ammonium ion was lower, 300 mM, in the presence of ADP than in the reaction without ADP (400 mM NH4+ ). The presence of strong substrate inhibition and non-hyperbolic kinetics did not allow the use of the standard Lineweaver-Burk plot or even the Hill plot in order to determine kinetic constants. Both plots were curvilinear (not shown).

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JAN K, TELLER

,.x.,. 4

_

_

4

2

NH;

6

8

0

30

50

9 - oxoglutarate [mMl

lmi]

Fig. 1. The effect of N H ~ on the N A D H linked reaction o f G D H from mealworm fat body in the presence ( 0 ) and absence ( O ) o f 1 m M ADP. Activities were measured in 50 m M triethanolamine--HCI-NaOH buffer, p H 7.5, 10 m M 2-oxoglutarate and 0.2 m M N A D H .

Calculations performed according to the Kurganov method (Silonova et al., 1969) confirmed that the Hill equation was not valid in that case, especially using experimentally measured V=. Linearization of the Hill plot was made according to Endrenyi et aL (1975) (Table 1). The effect of varied concentrations of the second substrate, 2-oxoglutarate, was shown in Fig. 2. As for ammonium ion 1 mM ADP enhanced the activity at all substrate concentrations studied, with maximal effect of about 1.5-fold value at 10mM 2-oxoglutarate. Substrate inhibition occurred in both cases. The Hill plots constructed using experimentally measured Vm values were also strongly curvilinear, but the Lineweaver-Burk plots were linear up to V~ (not shown). The effect of the third substrate, NADH, is shown in Fig. 3. Maximal activation by 1 mM ADP was observed at 0.2mM NADH. Substrate inhibition also occurred. The standard Hill plots were parallel and essentially linear. The determined kinetic constants for all substrates are presented in Table 1. Activation of G D H by ADP at different concentrations of the effector was also measured in optimal conditions (Fig. 4). ADP enhanced the rate of N A D H oxidation by about 50%. Activation constant, Ka, determined from the modified Hill plot (inset in the Fig. 4) was equal to 0.13 mM and h = 1.88. G D H activity was also measured in different corn-

Fig. 2. The effect of 2-oxoglutarate on the N A D H linked reaction o f G D H from mealworm fat body in the presence ( 0 ) and absence ( O ) of I m M ADP. Activities were measured in 50 m M t r i e t h a n o l a m i n e - H C I - N a O H buffer, p H 7.5, 0.2 m M N A D H and 300 m M NH4+ in the presence or 400 m M NH4+ in the absence o f ADP.

binations of concentrations of all three substrates in the absence or presence or ADP. The values of/(o.s and h for ammonium ion are shown in Tables 2 and 3. The K0.5 values varied from 72 to 254 mM, and h from 1.6 to 2.2 in the absence of ADP. In the presence

./" 0

I

I

0 5

0.'~, NADH

~.3

o.5

[raM]

Fig. 3. The effect o f N A D H on the N A D H linked reaction o f G D H from meaiworm fat body in the presence ( 0 ) and absence ( O ) of 1 m M ADP. Activities were measured in 50 m M triethanolamine-HC1-NaOH buffer, p H 7.5, 10 m M 2-oxoglutarate and 3 0 0 m M N H ~ in the presence or 400 m M NH4+ in the absence o f ADP.

Table 1. Kinetic constants for the NADH linked reaction of GDH from mealworm fat body NH +

lJll I I

Substrate 2-oxoglutarate

NADH

Activator /(0.5 h Vm K0.5 h Vm Ka.5 h Vm -ADP 201 1.79 1.65 2.58 1.00 1.63 0.054 1.49 1.64 + 1 mM ADP 65 1.35 2.36 3.20 1.00 2.36 0.067 1.49 2.40 Results are means of at least three determinations. K0.s and I'm values are in mM and t4kat/mg protein respectively.

Tenebrio glutamate dehydrogenase

about 90% after addition of the equimolar amounts of EDTA. Amino acid L-leucine activated the enzyme. About 20% activation was observed when the reaction was measured in the presence of 10mM leucine. Activation constant was equal to 2.5 mM; 10 mM leucine activated to the same extent oxidation of N A D H in the presence of 1 mM ADP as well as the reduction of NAD. In the same assay conditions bovine liver G D H was activated by 16%.

.,

,

, 05

,

331

I 'n

II

2 io

ADP [mM] Fig. 4. A D P activation of N A D H linked reaction of G D H from mealworm fat body. Velocity o f the reaction in the absence of A D P (v), which was taken as 100%, was subtracted from velocity in the presence of A D P (v~). Inset shows the modified Hill plot. Assay conditions were: 50 m M triethanolaminv--HCI-NaOH buffer, p H 7.5, 300 mM N H ~ , 0.2 mM N A D H and 10 mM 2-oxoglutarate.

of the activator K0.s values were lower and varied from 20 to 79 mM of NH~', and h varied from 1.0 to 1.3. Substrate inhibition was as strong as concentrations of N A D H and 2-oxoglutarate were low. The action of several well-known effectors on G D H activity was also studied. In optimal conditions without ADP the enzyme was activated by EDTA. Maximal activation of 22% was reached at the concentration of 0.25 mM EDTA. Zinc ions inhibited the enzyme strongly. The inhibition constant was equal to 0.055 mM. This inhibition was reversible in

N A D P H linked activity The effects of different concentrations of substrates and activator, ADP, were also investigated in the reaction of reductive amination with N A D P H as a coenzyme. Similar kinetic behaviour was observed when N A D H was a cocnzyme. The concentrations of substrates required for saturation of the enzyme were lower for NH~" and 2-oxoglutarate but almost identical for the coenzyme. Kinetic constants were as follows: K0.5 for ammonium ion was 62.5mM (h = 1.8), K0.5 for 2-oxoglutarate was 0.92mM (h = 1.21) and K0.s for NADPH was 0.058mM (h = 1.62). At the saturating concentrations of substrates, ADP activated the enzyme by the factor of 1.5 at concentrations of above 0.1 mM. Activation constant was 0.049 mM. DISCUSSION

Activity of the mealworm fat body G D H depends on the cocnzyme used. Reaction with N A D P H with and without ADP was I0 times slower than with NADH. High activity in the direction of N A D H oxidation is a common propi~rty of animal GDHs (Smith et al., 1975), including insect enzyme (Bond and Sang, 1968; Donnellan et al., 1974; Bursell, 1975; Male and Storey, 1982, 1983; Prezioso et al., 1985). Some authors argued that relatively high activity of G D H with NADPH as a cocnzyme indicates the Table 2. Ammonium ion kinetic constants for the NADH linked participation of the enzyme in the biosynthesis of reaction of GDH from mealworm fat body at different concenamino acids (Sies et al., 1974, 1975; Smith et al., 1975; trations of NADH and 2-oxoglutarate, and in the absence of ADP Tischler et al., 1977; Male and Storey, 1982). It seems NADH (raM) obvious that a similar pathway of amino acid bio2-oxoglutarate (raM) 0.05 0.075 0.1 0.15 0.2 synthesis occurs in insects, ¢speciaUy in the larval fat 1.5 ~.s 86 1~ 72 86 89 body and G D H takes part in this process. h 1.7 1.8 1.8 1.6 1.9 Affinity constants determined in the reductive ami5.0 ~.5 1~ !18 101 139 139 nation reaction should be examined from several h 1.9 1.6 1.9 1,8 i.9 points of view. Comparison with affinity constants 10.0 K°'s 169 155 212 254 201 h 2.0 1.6 2.1 2.2 1.8 for other animal GDHs may indicate different, even Results are means of at least three determinations. The values of K0.s unique properties of the fat body GDH. Thus, are in raM. for GDHs from vertebrate tissues (mammals, birds, amphibians and fish) substrate constants are (mM): NADH, 0.02-0.04; NADPH, 0.02-0.04; Table 3. Ammonium ion kinetic constants for the NADH linked 2-oxoglutarate, 0.2-5.0 and ammonium ions, 0.5-105 reaction of GDH from mealworm fat body at different concen(Smith et al., 1975; Chee et al., 1979; Prezioso et al., trations of NADH and 2-oxoglutarate, and in the presence of I mM 1985). For insect GDHs those constants are (mM): ADP NADH, 0.01-0.045; NADPH, 0.04-0.07; 2-oxogluNADH (raM) tarate, 0.26-3.3 and ammonium ions, 11-600(Bond 2-oxoglutarat¢ (mM) 0.05 0.075 0.1 0.15 0.2 and Sang, 1968; Bursell, 1975; Male and Storey, 1982, 1983; Prezioso et al., 1985). As can be seen the values 1.5 K°.s 40 22 20 43 30 h 1.1 1.0 1.0 1.0 1.3 of affinity constants for vertebrate and insect enzymes K0.s 55 40 26 61 56 are very similar, for some substrates even identical. 5.0 h 1.1 1.0 1.0 !.0 1.1 Only values for ammonium ions differ markedly. K0.s 58 52 41 79 65 10.0 h 1.2 i.1 1.0 1.0 1.3 Affinity constants for ammonium ions are very high Results are means of at least three determinations. The values of K0.s for insect enzyme. Such high K0.s, up to several are in mM. hundred mM, are not common even for micro-

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organism and plant GDHs (Shatilov, 1982). Saturation of Tenebrio GDH with ammonium ion was reached only at 300-400 mM. ADP as an activator increases not only maximal velocities, but also affects the values of substrate constants at saturating concentrations of cosubstrates. Addition of ADP increased K0.5 for 2-oxoglutarate by 1.2-fold, but decreased K0.s for NADH by 0.8-fold and K0.5 for ammonium ions by 0.3-fold. These changes were similar to those observed by Male and Storey (1982) for Eurosta GDH. Kinetic behaviour of G D H in the reductive amination reaction with NADH as a coenzyme at different substrates concentrations suggests that the enzyme can function in very different metabolic conditions. At saturating concentrations of the two substrates G D H had low affinity for the third substrate, but a fall in N A D H and 2-oxoglutarate concentrations decreased drastically affinity constants for ammonium ions. The presence of 1 mM ADP increased GDH activity and decreased affinity constants. ADP also decreased cooperativity coefficients: reaction in the presence of ADP showed hyperbolic kinetics. Purified Tenebrio GDH was activated by EDTA. This effect was a result of the interaction of EDTA with the enzyme, but not of the removal of bivalent cations as in the case of crude preparations. Zinc ions at the concentration of 1 mM inhibited the enzyme completely, but reversibly. The majority of GDHs are activated by relatively high concentrations (about 10raM) of L-leucine (Goldin and Frieden, 1971). The effect of this amino acid on insect G D H has been investigated by Male and Storey (1983). These authors did not detect any effect of leucine on Popilla japonica GDH. In this study about 20% activation of Tenebrio GDH was reached at 10 mM leucine. The GDH activation by leucine in the mammalian liver has been investigated because it also inhibits urea synthesis and gluconeogenesis (McGivan and ChappeU, 1975; Rognstad, 1977). Elucidation of a possible role of leucine in the activation of insect G D H needs further studies. It should be noted here that leucine inhibits effectively another insect mitochondrial enzyme, i.e. alanine aminotransferase (Schneider and Chen, 1981). Kinetic properties of the purified Tenebrio G D H indicate that the enzyme can play a key role in the nitrogen and energy metabolism in the fat body. Its function is probably strongly dependent on a metabolic state of the tissue and can be regulated precisely by the energy charge and through the availability of substrates. It is also concluded from kinetic studies of glutamate deamination reaction (Teller, 1988). Specificity of larval metabolism suggests the anabolic function of G D H in the fat body. Synthesized glutamate would be utilized in various metabolic pathways, i.e. protein synthesis, synthesis of uric acid, in the maintenance of high level of glutamate in the haemolymph or in proline synthesis (Chert, 1966; Florkin and Jeuniaux, 1974; Bursell, 1977; Wyatt, 1980; Beenakkers et al., 1984). On the other hand, it seems also evident that the physiological state of the fat body can determine the direction of G D H reaction.

REFERENCES

Beenakkers A. M. Th., Van der Horst D. J. and Marrewijk W. J. A. (1984) Insect flight muscle metabolism. Insect Biochem. 14, 243-260. Bond P. A. and Sang J. H. (1968) Glutamate dehydrogenase of Drosophila larvae. J. Insect Physiol. 14, 341-359. Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Bursell E. (1975) Glutamic dehydrogenase from sarcosomes of the tsetse fly (Glossina morsitans) and the blowfly (Sarcophaga nodosa). Insect Biochem. 5, 289-297. Bursell E. (1977) Synthesis of proline by fat body of the tsetse fly (Glossina morsitans). Metabolic pathways. Insect Biochem. 7, 427-434. Caggese C., DePinto V. and Ferrandino A. (1982) Purification and genetic control of NAD-dependent glutamate dehydrogenase from Drosophila melanogaster. Biochem. Genet. 20, 449-460. Chee P. Y., Dahl J. L. and Fahien L. A. (1979) The purification and properties of rat brain glutamate dehydrogenase. J. Neurochem. 33, 53--60. Chen P. S. (1966) Amino acid and protein metabolism in insect development. In Advances in Insect Physiology (Edited by Beament J. W. L., Treherne J. E. and Wigglesworth V. B.), Vol. 3, pp. 53-132. Academic Press, London. Donnellan J. F., Jenner D. W. and Ramsey A. (1974) Subcellular fractionation of fleshfly flight muscle in attempts to isolate synaptosomes and to establish the location of glutamate enzymes. Insect Biochem. 4, 243-265. Endrenyi L., Fajszi C. and Kwong F. H. F. (1975) Evaluation of Hill slopes and Hill coefficientswhen the saturation binding or velocity is not known. Eur. J. Biochem. 51, 317-328. Florkin M. and Jeuniaux C. (1974) Hemolymph composition. In The Physiology of Insecta (Edited by Rockstein M.), Vol. 5, pp. 317-473. Academic Press, London. Goldin B. R. and Frieden C. (1971) L-glutamate dehydrogenases. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. R.), Vol. 4, pp. 77-117. Academic Press, New York. McGivan J. D. and ChappeU J. B. (1975) On the metabolic function of glutamate dehydrogenase. FEBS Lett. 52, I-7. Male K. B. and Storey K. B. (1982) Purification and properties of glutamate dehydrogenase from cold-hardy gall fly larva, Eurosta solidaginis. Insect Biochem. 12, 507-514. Male K. B. and Storey K. B. (1983) Tissue specific isozymes of glutamate dehydrogenase from the Japanese Beetle, Popillia japonica: catabolic vs anabolic GDH's. J. comp. Physiol. 151B, 199-205. Prezioso G., Indiveri C. and Bonvino V. (1985) Kinetic characterization of L-glutamate dehydrogenase isolated from Drosophila melanogaster larvae. Comp. Biochem. Physiol. 80B, 1-4. Rognstad R. (1977) Sources of ammonia for urea synthesis in isolated rat liver cells. Biochim. biophys. Acta 496, 249-254. Schneider M. and Chen P. S. (1981) L-alanine aminotransferase in Drosophila nigromelanica: isolation, characterization and activity during ontogenesis. Insect Biochem. 11, 657--673. Shatilov V. R. (1982) Glutamate dehydrogenases of microorganisms and plants. Uspekhi Biologhicheskoj Chimii 23, 185-209 (in Russian). Sies H., Haussinger D. and Grosskopf M. (1974) Mitochondrial nicotinamide nucleotide systems: ammonium chloride responses and associated metabolic transitions in

Tenebrio glutamate dehydrogenase hemoglobin-free perfused rat liver. Hoppe-Seyler's Z. physiol. Chem. 355, 305-318. Sies H., Summer K. H. and Biicher T. (1975) A process requiring mitochondrial NADPH: urea formation from ammonia. FEBS Lett. 54, 274-278. Silonova G. V., Livanova N. B. and Kurganov B. I. (1969) Allosteric inhibition of phosphorylase B from rabbit muscle. Molec. Biol. 3, 768-784 (in Russian). Smith E. L., Austen B. M., Blumenthal K. M. and Nyc J. F. (1975) Glutamate dehydrogenases. In The Enzymes (Edited by Boyer P. D.), Vol. XI, Part A, pp. 293-367. Academic Press, New York. Teller J. K. (1988) Purification and some properties of

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glutamate dehydrogenase from the mealworm fat body. Insect Biochem. (in press). Teller J. K. and Pilc L. (1985) Insulin in insects: analysis of immunoreactivity in tissue extracts. Comp. Biochem. Physiol. 81B, 493-497. Tischler M. E., Hecht P. and Williamson J. R. (1977) Effect of ammonia on mitochondrial and cytosolic NADH and NADPH systems in isolated rat liver cells. FEBS Lett. 76, 99-104. Wyatt G. R. (1980) The fat body as a protein factory. In Insect Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 201-225. Academic Press, New York.