Biochem. Physiol. Pflanzen 177, 537-540 (1982)
Inhibition of Wheat Succinic Semialdehyde Dehydrogenase by Oxaloacetate L. GALLESCHI, C. NOCCHI, C. FLORIS and G. BEDINI Institute of Botany, University of Pisa, Italy Key Term Index: succinic semialdehyde dehydrogenase, oxaloacetate, embryos; Triticum durum
Summary The effect of Krebs' cycle intermediates, nucleotides, aminoacids and polyamines on succinic semialdehyde dehydrogenase activity was studied. The enzyme is noncompetitively inhibited by oxaloacetate whereas other tested compounds have no effect. The Kj in the presence of succinic semialdehyde and NAD were determined. The results are discussed in relation to the y-aminobutyrate shunt.
Succinic semialdehyde dehydrogenase (EC 188.8.131.52; SSA-DH) catalyzes the oxidation of succinic semialdehyde to succinic acid. The enzyme is related to the y-aminobutyric acid metabolism (GABA-shunt; Fig. 1) either in bacteria (JAKOBY and SCOTT 1959) and plants (BALDY 1977) or in animals (DE BOER and BRUINVELS 1977). In higher plants, like Pisum, Phaseolus and Arachis, the final product of the pathway is metabolized by the mithocondrial enzymes of the Krebs' cycle (DIXON and FOWDEN 1961). This fact raises the possibility that some of intermediates of the cycle are able to modulate the enzyme activities of the GABA-shunt, as it has been demonstrated on the glutamate decarboxylase (GAD; BALDY 1975) and the SSA-DH (BALDY 1977) from Agaricus bisporus. In this paper the effects of Krebs' cycle intermediates, nucleotides, aminoacids and polyamines on the SSA-DH activity from durum wheat are reported. C02
Fig. 1. y-aminobutyrate shunt. GABA-T y-aminobutirate transaminase.
Table 1. Effect of different compounds (Krebs intermediates, chetoacids, aminoacids, polyamines and nucleotides) on SSA-DH activity. a)
oxaloacetate concentration mM 0.010 0.015 0.020 1.000
27.8 50.0 63.9 100.0
b) Tested compounds with no effect on SSA-DR activity: Acetyl-Co A, Cis aconitate, Citrate, Fumarate, Isocitrate, 2-Ketoglutarate, Malate, Phosphoenolpyruvate, Pyruvate, Succinate, y-aminobutyrate, Glutamate, Putrescine, Spermidine, Spermine, AMP, ADP, ATP Isolated embryos from dry durum wheat caryopses (Triticum durum cv Cappelli; 1 year old seeds, 100 % germination after 72 h) were utilized as enzymatic source. The SSA-DR was extracted and purified by means of the procedure devised by GALLESCHI et al. (1981 in press); the enzyme activity was measured spectrophotometrically as described by GALLESCHI et al. (1981 in press). The effectors, at 1 mM concentration (unless otherwise stated), were incubated with the enzyme for 3 min at 30 "C. 0.04
1 4 mM SSA • 10
Fig. 2. Effect of oxaloacctatc (20 f..lM) on the oxidation of 88A by 8SA-DH at variable concentrations of 8SA. /'" in the presence, 0 in the absence of the inhibitory.
Inhibition of Succinic Semialdehyde Dehydro genase
1 • 104 mM NAD Fig. 3. Effect of o.w/oacetate (20 pM)
oxidation of SSA by SSA-DH at variable cOllcentration of
N A D.
A in th e presence, •
in th e ab sence of th e inhib itor.
The Kj of oxaloacetate was calculat ed from the Lineweaver "- Burk plots of reciprocal initial reacti on velociti es against recipro cal substrate concent rations. Succinic sernialdehyde, aminoacids. nucleotides, polyamines and t ricarboxylic acid cycle intermediates were pur chased from Sigma Chemical Co., St. LOllis, Mo. U.S.A. Oth er reagent and buffer salts were reagent grade materials.
The effect of various compounds on SSA-DH activity at fixed concentrations of substrate, NAD, effectors and enzyme was evaluated. The results, recorded on Table 1, show that the oxaloacetate is the only one, among the effectors by us assayed, capable to determine a strong inhibition on SSA-DH activity. The oth er Krebs' cycle intermediates and the chetoaeids, as well as the assayed nueleotides, aminoacids and polyamines do not affect the SSA-DH activity. The inhibition of SSA-DI-I by oxaloacetate is apparently noncompetitive with the substrate at variable concentrations of SSA and NAD (Figs. 2 and 3). The values of th e inhibition constants of oxaloacetate were also det ermined to be 1.88 . 10-5111 in resp ect to SSA and 2.5 ·10-5ll-I in resp ect to NAD. The obtained results seem to elueidate some aspects about the little known regulatory prop erties of the GAB A-shllnt . Moreover the y app ear to be in some contrast with th e data obtain ed in oth er plan t materials. In Agaricus bisporus it has been demonstrated that SSA-D H, extracted from fruit-bodies, is noncomp etitively inhibited by malate
L. GALLESClII et aI., Inhibition of Suc cini c Semi aldehyde Dehydrogenase
(BALDY 1977): this one is not capable to exert any effect on durum wheat SSA-DH. No data exist about the inhibition by oxaloacetate on Agaricus bisporus SSA-DH activity which is noncompetitively inhibited by ADP and ATP (BALDY 1977). These nueleotides are ineffective on the wheat enzyme. The SSA-DH from both Agaricus and Triticum is not affectedby GABA or glutamate. Thepolyamines spermidine and spermine, which inhibitthe SSA-DH frommouse brain (LAPINJOKI et al. 1980), do not affect the wheat enzyme. From the presented data it appears that some differences in the regulation properties between the enzymes from Agaricus and Triticum exist. In the wheat caryopsis the SSA-DH is inhibited by oxaloacetate produced by malate dehydrogenase. In this way the oxidation ofSSA to succinic acid would be inhibited and so on this intermediate would be formed by the oxidation of£x-ketoglutarate. Therefore, it is possible that the inhibition of SSA-DH by oxaloacetate has a physiologic significance in the regulation ofthe GABA-shunt in plants. Acknowledgements Th e t echnical assistance of Mr. F. SAVIOZZI is gratefully a cknowledged. The work was supported by a National Research Council (C.N . R , ROME, Ital y) cont rac t.
References BALDY, P.: J.\Ietaholism e du y-a mino butyrate chez Agaricus bisporus, T. La L-glutamate-1-carboxylyase. Ph ysiol, Plant 34 , 365 - 372 (19 75). BALDY, P.: l\fetabolisme du y -a minobutyra te ch ez Agaricus bisporus. III. La succ ina t e-semialdehyde: NAD( P) + oxyrloreductase. Physiol. Plant 40, 91-97 (1977). D E BOER, TH., a nd BRUINVELS, J. : Assay a nd properties of 4-aminobutyri c-2-oxoglutaric a cid transa minase a nd succinic semia lde hy de deh ydrogena se in rat br ain t issue. J . Ne ur ochem. 28, 471 -478 (1977). DI XON, R. O. D., a nd FOWD EN, L.: y-a minob utyric acid metabolism in plants. Part 2. Metabolism in higher plants. Ann. Bo t. Sot . 25 , 513-530 (1961). GALJ,ESC III, 1., N oconr, C., FLORIS, C., BEDINI, G., ANGUILL ESI, l\f. C., a nd GRILLI, I. : Purification and prop erties of succini c: semi ald ehyde dehydrogenase. Plant Cell Re port (in press) 1982. JAKOllY, W. B., and SCOTT, E. M.: Ald ehyde oxidation. III. Su ccinic semialdehyde dehydrogenase. .J. Biol, Chem. 234, 937- 940 (1959). LAI'INJOKI, S. P., PAJUNEN, A. E. I., HIETALA, O. A., und PIHA, R. S.: The effect of polyamines on the enzy mes of the 4-aminobutyri e aci d metabolism in mouse brain in vitro. FEBS Lett. 112, 289-292 (1980).
Received Ja nua ry 19,1982 Aut hors ' address: L. GALLESC III, C. NO('('HI, C. FLORIS and G. B EDI"'I, Institute of Botany, University of Pisa , I tal }".