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In vitro inhibition of yeast phenylalanyl-tRNA synthetase by ochratoxin A
Chem. -Biol. Interactions, 24 (1979) 257-261 o Rl~ev~er~North-Hoi~a~d Scientific Publishers Ltd.
257
Short Communication IN VITRO ENHIBITION OF YEAST PHENYLALANYL-tRNA SYNTHETASE BY OCHRATOXIN A E.-E. CREPPY, A.A.J. LUGNIER, and C. DIRHEIMER
F. FASlOLO,
R. HELLERa, R. R~S~HENTHALER~
Laboratoire de Toxicologic et de Bioiogie Mole’culaires, Faculte’ de Pharmacie, Universit& Louis Pasieur, et I,B.L%KC. du CNflS, 15 rue lkscarie~~ ~~~~~0~~~(Fmnce:e) and ak.&iCuZ f&r Mikrobiologie, Universiflit Mt‘ilaster. Westphalen (G.F.R.) (Received September 5th, 1978) (Accepted Octaber 26th, 1978)
Introduction
Qchratoxin A (OT-A) is a mycotoxin, produced by ubiquitous molds. It causes essentially liver and kidney damages in animals [l] _ Gont~i~at~un of cereals by the toxin have been reported [If. Et is composed of a phenylalanine moiety bound by o-amide bond to a dihydroisocoumarin chlorinated in the 5’ position. OT-A has antibiotic properties on gram-positive species [ 23. Its mechanism of action has been studied in vitro with bacterial systems [ 3,4] . Using a partially purified phenylalanyl-tRNA synthetase from Bacillus subtiks, Konrad and Raschenthaler [ 3J showed that QT-A acts as a competitive inhibitor with respect to phenylalan~e in the tRNAPhe acylation. With an in vitro poly-U directed polyphenylalanine synthesizing system of Bacillus stearotherrnophilus a similar competitive inhibition was shown 141, but no inhibition was observed when phenylalanyl-tRNA isntead of phenylalanine was used as substrate for peptide synthesis. These results show that the target of OT-A inhibition in bacteria is phenylalanyl-tRNA synthetase. As OT-A is highly toxic for higher organisms [l] it was also interesting to test its action on eukaryotic phenylalanyl-tRNA synthetase (PheRS) and to study nut only its action on the overall tRNA aminoacyia.tion bus also on the phenylalanine activation i.e. the first step of the acylation reaction catalysed by the enzyme. Materials and Methods
OT-A was isolated and purified from wheat kernels infected by Aspergillus as previously described (2). [ “4C]phenylalanine and f 32P]pyrophosphate were from C.E.A., Saclay, France. Yeast phenyl~a~yl-tR~~~ synthetase (PheRS) was prepared according to Fasiolo et al. [ 51. It was electrophoretically pure and had a specific activity of 3200 unitsjmg, Enzyme units are defined as described in f5] , Pure yeast phenylalani~e tRNA was prepared by counter-current distribution according to Dirheimer and Ebel [6]. All other chemicals and reagents were either analytical or reagent grade.
ochraeeus
OT-A concentration (mg/ml) was determinated by U.V. absorbance at 332 and 213 nm, with E 6400 and 35 800 respectively. The phenylalanine activation was assayed by the [32P]pyrophosphate-ATP exchange using the conditions described by Gangloff et al. [ 71. The reaction mixture (100 ~1) contained ATP 2 mM, [32P]pyrophosphate (spec. act., 28 mCi/mM) 2 mM, Tris-HCl pH 7.8 144 mM, MgC1210 mM, p-mercaptoethano1 5 mM, PheRS 5.6 nM, and variable amounts of phenylahmine and OT-A as indicated. After preincubation of the reaction medium at 37’C, OT-A was added followed by the enzyme. After 10 min of incubation the reaction was stopped by the addition of 100 ~1 of a 7% perchloric acid solution and 0.5 M pyrophosphate. The radioactive ATP was adsorbed on activated charcoal (200 ~1 of a 3% water suspension). The charcoal was filtered on glass fiber filters (GF/C Whatman) which were washed with 5% trichloroacetic acid and dried. The radioactivity was counted in an Intertechnique scintillation counter in the presence of 5 ml 0.45% Omnifluor in toluene. A time dependence curve was made which showed that the pyrophosphate-ATP exchange reaction was linear at least for the first 12 min after starting the reaction. The tRNA aminoacylation mixture (350 ~1) contained tRNAPhe 6 MM, ATP 10 mM, PheRS 0.76 nM, MgC12 15 mM, Tris-HCl pH 7.8 144 mM, reduced ghrtathione 2 mM, bovine serum albumin 1.5 PM, variable amounts of [ 14C]phenylalanine (spec. act., 32 mCi/mM), and OT-A. After incubation of this reaction mixture at 25°C the enzyme was added. Aliquots of 80 ~1 were withdrawn at 1 min intervals and put on Whatman 3MM paper discs,
Fig. 1. Dxan plot of phenylalanine dependent reaction c&alyze’d by yeast PheRS. ao, 0.01 mM of phenylalanine.
[3aP]pyrophosphate-ATP mM of phenylalanine;
q
-
exchange 0, 0.1
259 cpm X
lo-’
.
0.8
I
I
I
I
1
2
3
4
b
tnin
Fig. 2. Kinetic of tRNAPhe aminoacylation catalyzed by yeast PheRS with 5 NM of [‘4C]phenylalanine and 4.5 nM enzyme concentration. O---=-Q, control; o0, 1.5 mM ,3 mM OT-A;a------A, 2 mM OT-A. OT- A ; .
which were treated according to the technique Finally the radioactivity of the discs was counted
of Mans and Novelli [8]. as above.
Results The phenylalanine-dependent [ “P]pyrophosphate-ATP exchange reaction, catalyzed by pure yeast phenylalanyl-tRNA synthetase, was shown to be inhibited by OT-A. The initial reaction velocities (v) were determined as a function of the OT-A concentrations. The kinetics were done in triplicate. The inhibition constant (Ki), obtained from a Dixon plot of the reaction, was 1.5 mM (Fig. 1) whereas the Michaelis constant (Km) of the enzyme for phenylalanine was found to be 0.03 mM [ 51. Inhibition by OT-A of tRNA acylation by phenylalanine catalyzed by the same enzyme was also followed. The reaction velocity was lowered in presence of OT-A (Fig. 2). Kinetics by tRNAPhe charging were pe rformed with different phenglalanine concentrations in the presence of ochratoxin. The initial velocities obtained were used to draw the curves in Fig. 3. They show that OT-A competitively inhibits the reaction with a Ki of 1.5 mM whereas the Km of phenylalanine is 5 PM.
Conclusions As in bacterial
systems,
OT-A acts as a competitive
inhibitor
of phenyl-
$- (cpm X
lO_‘) 5 PM Phe /
/lO
PM Phe
Phe
i ;_____ -Ki
0.5
1
Fig. 3. Dixon plot of tRNA 5 PM phenylalanine; ~3-0, nM enzyme concentration.
1.5
2
3
OT-A
mM
acylation of phenylalanine 10 MM phenylalanine; A-
catalyzed by PheRS. w---= *, 50 PM phenylalanine; 4.;
alanine, in both the yeast phenylalanyl-tRNA synthetase-catalyzed pyrophosphate-ATP exchange and the tRNA Phe charging reaction. But the affinity of the pure enzyme for the toxin is rather low: 1.5 mM whereas its affinity for phenylahmine is 300 times greater in the overall reaction. With Bacillus subtilis phenylalanyl-tRNA synthetase Konrad and RGschenthaler [3] found that the Ki is only about 2 times higher than the K, (1.3 PM). This difference could mean that OT-A is much more toxic to bacteria than to higher cells. Heller et al. [2] showed that on Streptococcus fueculis a 37 r.lM concentration of OT-A has a bacteriostatic action. Natori et al. [9] , and Arai and Otomo [lo) showed that a 77 PM concentration of OT-A has a cytotoxic action on Hela cells. Thus the OT-A concentrations active on whole organism from the two different kingdoms are much closer than the inhibitory concentrations on the target enzyme. The results also show that at a same concentration of phenylalanine and ochratoxin the exchange reaction is more inhibited than the aminoacylation of the tRNA. This is explained by the 5 times lower Km for phenylalanine of
261 the aminoacylation reaction as compared to the exchange reaction. The degree of inhibition is in fact related by the following expression: % inhibition =
I I+Ki(l++) m
where I is the concentration of OT-A and S the concentration of phenylalanine. The results demonstrate for the first time that OT-A inhibits an enzyme essential for protein biosynthesis in higher organisms. It remains to be explained why, although it has an affinity for the PheRS 300 fold lower than the substrate, it has such high toxicity [ 1). We thank Dr. J. Bonnet for helpful discussion and Mrs M. Schlegel who did the counter-current distribution of tRNA. This work was supported by grants from the Institut National de la Sante et de la Recherche Mddicale (Contrat de Recherche Libre INSERM 76.10.613) and from the DQpartement de Biologid du Commissariat a 1’Energie Atomique. 1 J. Harwig, Ochratoxin A and related metabolites, in: I.F.H. Purchase (Ed.), Mycotoxins, Elsevier Scientific Publishing Company, Amsterdam, Oxford, New York, 1974, pp. 345-367. 2 K. Heller, C. Schultz, A. Loser and R. RSschenthaler, Can. J. Microbial., 21 (1975) 972. 3 I. Konrad and II. R&chenthaIer, FEBS L&t., 83 (1977) 341. 4 I. Bunge, G. Dirheimer and R. RBschenthaler, Biochem. Biophys. Res. Commun., 83 (1978) 398. 5 F. Fasiolo, N. B&fort, Y. Boulanger and J.P. Ebel, Biochim. Biophys. Acta, 217 (1970) 305. 6 G. Dirheimer and J.P. Ebel, Bull. Sot. Chim. Biol., 49 (1967) 1679. 7 J. Gangloff, A. Schutz and G. Dirheimer, Eur. J. Biochem., 65 (1976) 177. 8 R.J. Mans and G.D. Novelli, Arch. Biochem. Biophys., 94 (1961) 48. 9 S. Natori, F. Sasaki, H. Kurata, S. Udagawa, M. Ichinoe, .XI. Saito and M. Umeda, Chem. Pharm. Bull., 18 (1970) 2259. 10 T. Arai and M. Otomo, Ann. Rept. Int. Fd Microbial., 22 (1969) 81.
257
Short Communication IN VITRO ENHIBITION OF YEAST PHENYLALANYL-tRNA SYNTHETASE BY OCHRATOXIN A E.-E. CREPPY, A.A.J. LUGNIER, and C. DIRHEIMER
F. FASlOLO,
R. HELLERa, R. R~S~HENTHALER~
Laboratoire de Toxicologic et de Bioiogie Mole’culaires, Faculte’ de Pharmacie, Universit& Louis Pasieur, et I,B.L%KC. du CNflS, 15 rue lkscarie~~ ~~~~~0~~~(Fmnce:e) and ak.&iCuZ f&r Mikrobiologie, Universiflit Mt‘ilaster. Westphalen (G.F.R.) (Received September 5th, 1978) (Accepted Octaber 26th, 1978)
Introduction
Qchratoxin A (OT-A) is a mycotoxin, produced by ubiquitous molds. It causes essentially liver and kidney damages in animals [l] _ Gont~i~at~un of cereals by the toxin have been reported [If. Et is composed of a phenylalanine moiety bound by o-amide bond to a dihydroisocoumarin chlorinated in the 5’ position. OT-A has antibiotic properties on gram-positive species [ 23. Its mechanism of action has been studied in vitro with bacterial systems [ 3,4] . Using a partially purified phenylalanyl-tRNA synthetase from Bacillus subtiks, Konrad and Raschenthaler [ 3J showed that QT-A acts as a competitive inhibitor with respect to phenylalan~e in the tRNAPhe acylation. With an in vitro poly-U directed polyphenylalanine synthesizing system of Bacillus stearotherrnophilus a similar competitive inhibition was shown 141, but no inhibition was observed when phenylalanyl-tRNA isntead of phenylalanine was used as substrate for peptide synthesis. These results show that the target of OT-A inhibition in bacteria is phenylalanyl-tRNA synthetase. As OT-A is highly toxic for higher organisms [l] it was also interesting to test its action on eukaryotic phenylalanyl-tRNA synthetase (PheRS) and to study nut only its action on the overall tRNA aminoacyia.tion bus also on the phenylalanine activation i.e. the first step of the acylation reaction catalysed by the enzyme. Materials and Methods
OT-A was isolated and purified from wheat kernels infected by Aspergillus as previously described (2). [ “4C]phenylalanine and f 32P]pyrophosphate were from C.E.A., Saclay, France. Yeast phenyl~a~yl-tR~~~ synthetase (PheRS) was prepared according to Fasiolo et al. [ 51. It was electrophoretically pure and had a specific activity of 3200 unitsjmg, Enzyme units are defined as described in f5] , Pure yeast phenylalani~e tRNA was prepared by counter-current distribution according to Dirheimer and Ebel [6]. All other chemicals and reagents were either analytical or reagent grade.
ochraeeus
OT-A concentration (mg/ml) was determinated by U.V. absorbance at 332 and 213 nm, with E 6400 and 35 800 respectively. The phenylalanine activation was assayed by the [32P]pyrophosphate-ATP exchange using the conditions described by Gangloff et al. [ 71. The reaction mixture (100 ~1) contained ATP 2 mM, [32P]pyrophosphate (spec. act., 28 mCi/mM) 2 mM, Tris-HCl pH 7.8 144 mM, MgC1210 mM, p-mercaptoethano1 5 mM, PheRS 5.6 nM, and variable amounts of phenylahmine and OT-A as indicated. After preincubation of the reaction medium at 37’C, OT-A was added followed by the enzyme. After 10 min of incubation the reaction was stopped by the addition of 100 ~1 of a 7% perchloric acid solution and 0.5 M pyrophosphate. The radioactive ATP was adsorbed on activated charcoal (200 ~1 of a 3% water suspension). The charcoal was filtered on glass fiber filters (GF/C Whatman) which were washed with 5% trichloroacetic acid and dried. The radioactivity was counted in an Intertechnique scintillation counter in the presence of 5 ml 0.45% Omnifluor in toluene. A time dependence curve was made which showed that the pyrophosphate-ATP exchange reaction was linear at least for the first 12 min after starting the reaction. The tRNA aminoacylation mixture (350 ~1) contained tRNAPhe 6 MM, ATP 10 mM, PheRS 0.76 nM, MgC12 15 mM, Tris-HCl pH 7.8 144 mM, reduced ghrtathione 2 mM, bovine serum albumin 1.5 PM, variable amounts of [ 14C]phenylalanine (spec. act., 32 mCi/mM), and OT-A. After incubation of this reaction mixture at 25°C the enzyme was added. Aliquots of 80 ~1 were withdrawn at 1 min intervals and put on Whatman 3MM paper discs,
Fig. 1. Dxan plot of phenylalanine dependent reaction c&alyze’d by yeast PheRS. ao, 0.01 mM of phenylalanine.
[3aP]pyrophosphate-ATP mM of phenylalanine;
q
-
exchange 0, 0.1
259 cpm X
lo-’
.
0.8
I
I
I
I
1
2
3
4
b
tnin
Fig. 2. Kinetic of tRNAPhe aminoacylation catalyzed by yeast PheRS with 5 NM of [‘4C]phenylalanine and 4.5 nM enzyme concentration. O---=-Q, control; o0, 1.5 mM ,3 mM OT-A;a------A, 2 mM OT-A. OT- A ; .
which were treated according to the technique Finally the radioactivity of the discs was counted
of Mans and Novelli [8]. as above.
Results The phenylalanine-dependent [ “P]pyrophosphate-ATP exchange reaction, catalyzed by pure yeast phenylalanyl-tRNA synthetase, was shown to be inhibited by OT-A. The initial reaction velocities (v) were determined as a function of the OT-A concentrations. The kinetics were done in triplicate. The inhibition constant (Ki), obtained from a Dixon plot of the reaction, was 1.5 mM (Fig. 1) whereas the Michaelis constant (Km) of the enzyme for phenylalanine was found to be 0.03 mM [ 51. Inhibition by OT-A of tRNA acylation by phenylalanine catalyzed by the same enzyme was also followed. The reaction velocity was lowered in presence of OT-A (Fig. 2). Kinetics by tRNAPhe charging were pe rformed with different phenglalanine concentrations in the presence of ochratoxin. The initial velocities obtained were used to draw the curves in Fig. 3. They show that OT-A competitively inhibits the reaction with a Ki of 1.5 mM whereas the Km of phenylalanine is 5 PM.
Conclusions As in bacterial
systems,
OT-A acts as a competitive
inhibitor
of phenyl-
$- (cpm X
lO_‘) 5 PM Phe /
/lO
PM Phe
Phe
i ;_____ -Ki
0.5
1
Fig. 3. Dixon plot of tRNA 5 PM phenylalanine; ~3-0, nM enzyme concentration.
1.5
2
3
OT-A
mM
acylation of phenylalanine 10 MM phenylalanine; A-
catalyzed by PheRS. w---= *, 50 PM phenylalanine; 4.;
alanine, in both the yeast phenylalanyl-tRNA synthetase-catalyzed pyrophosphate-ATP exchange and the tRNA Phe charging reaction. But the affinity of the pure enzyme for the toxin is rather low: 1.5 mM whereas its affinity for phenylahmine is 300 times greater in the overall reaction. With Bacillus subtilis phenylalanyl-tRNA synthetase Konrad and RGschenthaler [3] found that the Ki is only about 2 times higher than the K, (1.3 PM). This difference could mean that OT-A is much more toxic to bacteria than to higher cells. Heller et al. [2] showed that on Streptococcus fueculis a 37 r.lM concentration of OT-A has a bacteriostatic action. Natori et al. [9] , and Arai and Otomo [lo) showed that a 77 PM concentration of OT-A has a cytotoxic action on Hela cells. Thus the OT-A concentrations active on whole organism from the two different kingdoms are much closer than the inhibitory concentrations on the target enzyme. The results also show that at a same concentration of phenylalanine and ochratoxin the exchange reaction is more inhibited than the aminoacylation of the tRNA. This is explained by the 5 times lower Km for phenylalanine of
261 the aminoacylation reaction as compared to the exchange reaction. The degree of inhibition is in fact related by the following expression: % inhibition =
I I+Ki(l++) m
where I is the concentration of OT-A and S the concentration of phenylalanine. The results demonstrate for the first time that OT-A inhibits an enzyme essential for protein biosynthesis in higher organisms. It remains to be explained why, although it has an affinity for the PheRS 300 fold lower than the substrate, it has such high toxicity [ 1). We thank Dr. J. Bonnet for helpful discussion and Mrs M. Schlegel who did the counter-current distribution of tRNA. This work was supported by grants from the Institut National de la Sante et de la Recherche Mddicale (Contrat de Recherche Libre INSERM 76.10.613) and from the DQpartement de Biologid du Commissariat a 1’Energie Atomique. 1 J. Harwig, Ochratoxin A and related metabolites, in: I.F.H. Purchase (Ed.), Mycotoxins, Elsevier Scientific Publishing Company, Amsterdam, Oxford, New York, 1974, pp. 345-367. 2 K. Heller, C. Schultz, A. Loser and R. RSschenthaler, Can. J. Microbial., 21 (1975) 972. 3 I. Konrad and II. R&chenthaIer, FEBS L&t., 83 (1977) 341. 4 I. Bunge, G. Dirheimer and R. RBschenthaler, Biochem. Biophys. Res. Commun., 83 (1978) 398. 5 F. Fasiolo, N. B&fort, Y. Boulanger and J.P. Ebel, Biochim. Biophys. Acta, 217 (1970) 305. 6 G. Dirheimer and J.P. Ebel, Bull. Sot. Chim. Biol., 49 (1967) 1679. 7 J. Gangloff, A. Schutz and G. Dirheimer, Eur. J. Biochem., 65 (1976) 177. 8 R.J. Mans and G.D. Novelli, Arch. Biochem. Biophys., 94 (1961) 48. 9 S. Natori, F. Sasaki, H. Kurata, S. Udagawa, M. Ichinoe, .XI. Saito and M. Umeda, Chem. Pharm. Bull., 18 (1970) 2259. 10 T. Arai and M. Otomo, Ann. Rept. Int. Fd Microbial., 22 (1969) 81.