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Reversal of ethionine-induced inhibition of elongation of Avena coleoptiles by adenosine triphosphate
ARCHIVES
OF
BIOCHEMISTRY
Reversal
AND
BIOPHYSICS
108, 352-355 (1964)
of Ethionine-Induced
Avena
Coleoptiles
Inhibition
by Adenosine
W. E. NORRIS, From the Department
of Elongation
of
Triphosphate’
JR.2
of Biology, Southwest Texas State College, San Marcos, Texas Received
July
20, 1964
Ethionine inhibition of elongation of Avena coleoptiles has been measured in water and in 3-indoleacetic acid. In the presence of 0.01 M I-ethionine the inhibition amounts to about 70%. The application of adenosine triphosphate (ATP) effectively counteracts this inhibition; optimal ATP concentrations are between 2.5 and 5 X 1OW M. In the absence of ethionine ATP still exhibits a stimulatory effect on elongation. It is suggested that ethionine may exert its inhibiting effect on the protein synthesis necessary for elongation of the oat coleoptile by interference with ATP metabolism. INTRODUCTION
Ethionine, the ethyl analogue of methionine, has been widely used as a competitive inhibitor in studies of methionine metabolism, and it has been demonstrated that ethionine causes a loss of weight in rats (1). In fact, as pointed out by Shapiro and Schlenk (2), it may be termed the classical example of an antimetabolite. Among other things ethionine is known to inhibit (a) the formation of certain adaptive enzymes, (b) the lipotropic activity of methionine, (c) transmethylation, (d) formation of sulfurcontaining amino acids, or conversion of methionine to cystine, and (e) protein synthesis (3). The first studies of et,hionine effects on higher plants were those of Boll (4), who found that ethionine inhibition of the growth of excised tomato roots could be relieved by methionine, homocysteine, homocystine, and to a lesser degree by ethanolamine. Choline and glycine- betaine were ineffective. Ethionine and various ratios of 1 Supported by state appropriated funds for organized research to Southwest Texas State College. 2 This report was prepared at the University of Texas, Austin, during the summer of 1964, while the author was a National Science Foundation, Science Faculty Fellow. 352
methionine (or homocystine) to ethionine also exerted profound effects on the morphology of roots. S&rank (5) reported that both 3-indoleacetic acid (IAA) stimulated elongation of Avena coleoptile sections, and geotropic curvature of isolated coleoptiles with intact apices (endogenous auxin) were inhibited by 1-ethionine. The ethionine-induced inhibition of elongation was alleviated by 1-methionine and to a lesser degree by dlhomocysteine thiolactone-HCl, while neither choline-Cl nor methylmethioninesulfonium iodide were effective. L-Methionine was also effective in reversing the inhibition of geotropic curvature caused by ethionine. After exploring several possible explanations to account for the inhibitory effects of 1-ethionine, Schrank suggested that it might interfere with the utilization of methionine for protein syntheses which are required for elongation of the Avena coleoptile. Cleland (6) has investigated two possibilities of ethionine action in inhibiting elongation of Avma coleoptiles, that of blocking protein synthesis and inhibiting transmethylation. His data confirm Schrank’s observation of inhibition of IAAinduced elongation by ethionine and its reversal by methionine. Cleland separated
REVERSAL
OF ETHIONINE
total elongation of the coleoptile into reversible and irreversible components and found that ethionine caused an immediate inhibition of reversible elongation but did not inhibit irreversible elongation until 3-6 hours later. He suggested that inhibition of methylation was the cause of inhibition of reversible elongation, and that the inhibition of protein synthesis accounted for the inhibition of irreversible elongation. Farber (3) and Villa-Trevino et al. (7) call attention to the fact that the mechanism of the action of ethionine on protein synthesis is hazy, but is usually assumed to be some specific metabolic methionine-ethionine interaction, since methionine reverses most of the effects of ethionine. They note that clear-cut evidence supporting this is lacking, and since Shull (8) had observed that ethionine induced a marked decrease in liver ATP concentrat’ion shortly after its administration to rats, they investigated (7) the relationship of t’he reduced ATP level to the inhibition of protein synthesis induced by ethionine. Their experimental evidence shows that ethionine inhibits liver protein synthesis indirect’ly through an effect upon hepatic iZTP; this evidence supports an earlier suggestion of Stekol et al. (9).
The above cited experiments (7, 8), conducted primarily on rat liver, led to the work reported here, which indicates that ethionine may also exert it’s inhibitory effects on Arena coleoptile elongation by interference with ATP metabolism. MATERIALS
AND
METHODS
Buena sativa L. seeds, Victory strain (U. S. Department of Agriculture, C. I. 2020),3 were used in these experiments. The seedlings were grown on filter paper strips immersed in distilled water which previously had been aerated. Additional details of the growing method are described elsewhere (10). Only seedlings that were 72 hours old and that had 30 & 2 mm coleoptiles were used. Growth measurements were made on coleoptiles which were isolated from the seeds and primary leaves. The second 5.mm sections were used, measuring from the apex toward the base. These sections were floated in Petri dishes containing 3 Supplied culture.
by the C. S. Department
of Agri-
EFFECT
BY ATP
353
the media (20 sections/20 ml solution). All sections were randomized in distilled water and then floated on the experimental media. During cutting and transferring, the sections were exposed to low intensity red neon light (wavelengths longer than 6074 A), which previously had been observed to have no effect on the elongation of floating sections. The sections were allowed to elongate in the dark for 24 hours. At the end of this period, their lengths were measured with a micrometer in the ocular of a stereoscopic microscope. All procedures were carried out at a temperature of 22 rt 1°C. RESULTS
Figure 1 shows the effects of various concentrations of ATP on elongation of the oat coleoptile, both in water and 1-ethionine. The amount of elongation of the coleoptile segment in water is taken as the control value of lOO%, which, in a 24-hour period, is usually between 1.5 and 2.0 mm. It is apparent, from curve II that the addition of various concentrations of ATP resulted in a stimulation of elongation of the oat coleoptile amounting to a maximum of between 40 and 50% at an ATP concentrat’ion of 2.5 X 1O-4 M. Why higher concentrations of ATP inhibit elongation is not clear. When the coleoptile segments were allowed to elongate in 0.01 M 1-ethionine, an inhibition of approximately 70% resulted. Note that this line represents only 30% of the elongation shown in the water control. When ATP is added t,o the ethionine solution (curve I), the amount of inhibition is much reduced, e.g., an ATE’ concentration of 3.5 X 10-4M decreases the inhibition from 70% to between 10 and 15 %, that is to say, elongation in this case is 85-90% of the control value. The data presented in Fig. 2 differs only in that the control solution was 3-indoleacetic acid (0.1 mg per lit’er). In the presence of exogenous IAA the average elongation for a 24-hour period was about 3.5 mm as compared to 1.5-2.0 mm in wat’er. In general the results are the same as those shown in Fig. 1 except the stimulat’ing effect of ATP alone is not quite so marked. An enhancement of elongation of about 15 % resulted in ATP solutions of 1.25 and 2.5 X 1O-4 M, in IAA. Again 0.01 A// 1-ethionine inhibited elongation about 70%, and when ATP (2.5 X 1O-4 M) is added this is reduced to about 37%.
354
NORRIS
I
01
I 2
I
I 4
I 3
I 5
Cont.
of ATP
I 6
(X lO-4
I 7
I 8
I
I
5
10
Mob)
FIQ. 1. Influence of ATP on the elongation of the second 5-mm segment of the Avena coleoptile. (I) In a strongly inhibitory solution (0.01 M) of I-ethionine; (II) in water. Elongation is expressed as percentage of the water control. Standard deviations are shown. Each plotted point is the average of 1739 measurements. The figure represents the data of one typical experiment selected from a total of six replications. tions and partially reversing the ethionine inhibition of their elongation. This is true both in the presence and absence of exogenous IAA. From the preliminary
o
I
I I
I 2
I 3
Cont.
I 4
I 5
of ATP
(X d
I 6
I 7
I 8
Molar1
FIG. 2. Influence of ATP on the elongation of the second 5-mm segment of the Avena coleoptile
in IAA. (I) In a strongly inhibitory solution (0.01 M) of I-ethionine in IAA; (II) in IAA only. Elongation is expressed as percentage of the IAA control. Standard deviations are shown. Each plotted point is the average of 16-45 measurements. The figure represents the data of one typical experiment selected from a total of five replications. DISCUSSION It is evident from the data presented that the application of ATP is capable of stimulating the growth of Avena coleoptile sec-
work reported
here
no exact conclusions can be drawn as to the mechanisms involved. It may be recalled that Shull (8) noted that ethionine induced a marked decrease in the level of liver ATP, and in the later studies of Villa-Trevino et al. (7) this was observed to parallel the inhibition of protein synthesis. They also found that the administration of ATP or adenine counteracted both the inhibition of protein synthesis and the decreased level of hepatic ATP. These workers tested various compounds and found that only those convertible to adenine or an adenine derivative were effective, which led them to suggest that ethionine exerted its primary effect on the concentration of ATP and that the inhibition of protein synthesis was secondary. In an extensive discussion Villa-Trevino et al. (7) explored the possible manner in which ethionine affects the cellular level of ATP, and how adenine or ATP can counteract this effect, as well as the question as to how a decrease in ATP concentration inhibits the protein synthetic syst,em. They
REVERSAL
OF ETHIONINE
concluded that their data best support the idea detailed by Stekol et al. (9) and are supported by the work of Schmidt et al. (11) that ethionine exerts an adenine- or ATP-trapping effect. Briefly, based on Cantoni’s (12) original work, this involves the metabolic interaction of ATP with ethionine and methionine to form S-adenosylethionine and X-adenosylmethionine. Unlike the activated methionine, the X-adenosylethionine in effect represents a trap since the adenosine moiety is not readily released from this compound. With regard to the protein synthetic system, they suggest (7) that inhibition may be due to interference with the synthesis of “messenger RNA.” Webster (13) showed that ethionine blocks protein synthesis in plants, and both Schrank (5) and Cleland (6) suggested that the inhibitory effects of ethionine on elongation of the oat coleoptile might result from interference with protein synthesis. Nooden and Thimann (14) have recently presented evidence that some protein synthesis is required for auxin-induced cell enlargement. The above workers (5, 6, 13) did not specifically consider the manner in which inhibition of protein synthesis is brought about by ethionine, but the inference is on methionine-ethionine interaction. The data presented here are in agreement with the recently introduced concept that ethionine exerts its inhibitory effects by interference with ATP metabolism. The fact that ATP stimulates elongation of the oat coleoptile,
EFFECT
BY ATP
355
even in the absence of ethionine inhibition, might be taken as an indication that under normal conditions the metabolic energy supply (quantity of ATP) is not adequate to support maximal elongation. ACKNOWLEDGMENTS The technical assistance of Nick Vratis is acknowledged with pleasure. The author is indebted to Professor A. R. Schrank for criticism of the manuscript. REFERENCES 1. DYER, H. M., J. Biol. Chem. 134, 519 (1938). 2. SHAPIRO, S. K., AND SCHLENK, F., Advan. Enzymol. 22, 237 (1960). 3. FARBER, E., Advan. Cancer Res. 7, 383 (1963). 4. BOLL, W. G., Plant Phyeiol. 36, 115 (1960). 5. SCHRANK, A. R., Archiv. Biochem. Biophye. 61,
348 (1956). 6. CLELAND, It., Plant Physiol. 36, 5% (1960). 7. VILLA-TREVINO, S., SHULL, K. H., AND FARBER, E., J. Biol. Chem. 233, 1757 (1963). 8. SHULL, K. H., J. Biol. Chem. 237, PC1734 (1962). 9. STEKOL, J. A., ANDERSON, E. I., Hsu, P. T., AND WEISS, S., Abstr. 127th Meeting Am. Chem. SOL, Cincinnati, 1966~. 4C. American Chemical Society, Washington, D. C. 10. WIEGAND, 0. F., AND SCHRANK, A. R., Botan. Gaz. 121, 106 (1959). 11. SCHMIDT, G., SERAIDARIAN, K., GREENBAUM, L. M., HICKEY, M. D., AND THANNHAUSER, S. J., Biochim. Biophys. Acta 20, 135 (1956). 12. CANTONI, G. L., J. Biol. Chem. 189,745 (1951). 13. WEBSTER, G. C., Plant Physiol. 30, 351 (1955). 14. NOOD~N, L. D., AND THIMANN, K. V., Proc. Natl. Acad. Sci. U.S. 60, 194 (1963).
OF
BIOCHEMISTRY
Reversal
AND
BIOPHYSICS
108, 352-355 (1964)
of Ethionine-Induced
Avena
Coleoptiles
Inhibition
by Adenosine
W. E. NORRIS, From the Department
of Elongation
of
Triphosphate’
JR.2
of Biology, Southwest Texas State College, San Marcos, Texas Received
July
20, 1964
Ethionine inhibition of elongation of Avena coleoptiles has been measured in water and in 3-indoleacetic acid. In the presence of 0.01 M I-ethionine the inhibition amounts to about 70%. The application of adenosine triphosphate (ATP) effectively counteracts this inhibition; optimal ATP concentrations are between 2.5 and 5 X 1OW M. In the absence of ethionine ATP still exhibits a stimulatory effect on elongation. It is suggested that ethionine may exert its inhibiting effect on the protein synthesis necessary for elongation of the oat coleoptile by interference with ATP metabolism. INTRODUCTION
Ethionine, the ethyl analogue of methionine, has been widely used as a competitive inhibitor in studies of methionine metabolism, and it has been demonstrated that ethionine causes a loss of weight in rats (1). In fact, as pointed out by Shapiro and Schlenk (2), it may be termed the classical example of an antimetabolite. Among other things ethionine is known to inhibit (a) the formation of certain adaptive enzymes, (b) the lipotropic activity of methionine, (c) transmethylation, (d) formation of sulfurcontaining amino acids, or conversion of methionine to cystine, and (e) protein synthesis (3). The first studies of et,hionine effects on higher plants were those of Boll (4), who found that ethionine inhibition of the growth of excised tomato roots could be relieved by methionine, homocysteine, homocystine, and to a lesser degree by ethanolamine. Choline and glycine- betaine were ineffective. Ethionine and various ratios of 1 Supported by state appropriated funds for organized research to Southwest Texas State College. 2 This report was prepared at the University of Texas, Austin, during the summer of 1964, while the author was a National Science Foundation, Science Faculty Fellow. 352
methionine (or homocystine) to ethionine also exerted profound effects on the morphology of roots. S&rank (5) reported that both 3-indoleacetic acid (IAA) stimulated elongation of Avena coleoptile sections, and geotropic curvature of isolated coleoptiles with intact apices (endogenous auxin) were inhibited by 1-ethionine. The ethionine-induced inhibition of elongation was alleviated by 1-methionine and to a lesser degree by dlhomocysteine thiolactone-HCl, while neither choline-Cl nor methylmethioninesulfonium iodide were effective. L-Methionine was also effective in reversing the inhibition of geotropic curvature caused by ethionine. After exploring several possible explanations to account for the inhibitory effects of 1-ethionine, Schrank suggested that it might interfere with the utilization of methionine for protein syntheses which are required for elongation of the Avena coleoptile. Cleland (6) has investigated two possibilities of ethionine action in inhibiting elongation of Avma coleoptiles, that of blocking protein synthesis and inhibiting transmethylation. His data confirm Schrank’s observation of inhibition of IAAinduced elongation by ethionine and its reversal by methionine. Cleland separated
REVERSAL
OF ETHIONINE
total elongation of the coleoptile into reversible and irreversible components and found that ethionine caused an immediate inhibition of reversible elongation but did not inhibit irreversible elongation until 3-6 hours later. He suggested that inhibition of methylation was the cause of inhibition of reversible elongation, and that the inhibition of protein synthesis accounted for the inhibition of irreversible elongation. Farber (3) and Villa-Trevino et al. (7) call attention to the fact that the mechanism of the action of ethionine on protein synthesis is hazy, but is usually assumed to be some specific metabolic methionine-ethionine interaction, since methionine reverses most of the effects of ethionine. They note that clear-cut evidence supporting this is lacking, and since Shull (8) had observed that ethionine induced a marked decrease in liver ATP concentrat’ion shortly after its administration to rats, they investigated (7) the relationship of t’he reduced ATP level to the inhibition of protein synthesis induced by ethionine. Their experimental evidence shows that ethionine inhibits liver protein synthesis indirect’ly through an effect upon hepatic iZTP; this evidence supports an earlier suggestion of Stekol et al. (9).
The above cited experiments (7, 8), conducted primarily on rat liver, led to the work reported here, which indicates that ethionine may also exert it’s inhibitory effects on Arena coleoptile elongation by interference with ATP metabolism. MATERIALS
AND
METHODS
Buena sativa L. seeds, Victory strain (U. S. Department of Agriculture, C. I. 2020),3 were used in these experiments. The seedlings were grown on filter paper strips immersed in distilled water which previously had been aerated. Additional details of the growing method are described elsewhere (10). Only seedlings that were 72 hours old and that had 30 & 2 mm coleoptiles were used. Growth measurements were made on coleoptiles which were isolated from the seeds and primary leaves. The second 5.mm sections were used, measuring from the apex toward the base. These sections were floated in Petri dishes containing 3 Supplied culture.
by the C. S. Department
of Agri-
EFFECT
BY ATP
353
the media (20 sections/20 ml solution). All sections were randomized in distilled water and then floated on the experimental media. During cutting and transferring, the sections were exposed to low intensity red neon light (wavelengths longer than 6074 A), which previously had been observed to have no effect on the elongation of floating sections. The sections were allowed to elongate in the dark for 24 hours. At the end of this period, their lengths were measured with a micrometer in the ocular of a stereoscopic microscope. All procedures were carried out at a temperature of 22 rt 1°C. RESULTS
Figure 1 shows the effects of various concentrations of ATP on elongation of the oat coleoptile, both in water and 1-ethionine. The amount of elongation of the coleoptile segment in water is taken as the control value of lOO%, which, in a 24-hour period, is usually between 1.5 and 2.0 mm. It is apparent, from curve II that the addition of various concentrations of ATP resulted in a stimulation of elongation of the oat coleoptile amounting to a maximum of between 40 and 50% at an ATP concentrat’ion of 2.5 X 1O-4 M. Why higher concentrations of ATP inhibit elongation is not clear. When the coleoptile segments were allowed to elongate in 0.01 M 1-ethionine, an inhibition of approximately 70% resulted. Note that this line represents only 30% of the elongation shown in the water control. When ATP is added t,o the ethionine solution (curve I), the amount of inhibition is much reduced, e.g., an ATE’ concentration of 3.5 X 10-4M decreases the inhibition from 70% to between 10 and 15 %, that is to say, elongation in this case is 85-90% of the control value. The data presented in Fig. 2 differs only in that the control solution was 3-indoleacetic acid (0.1 mg per lit’er). In the presence of exogenous IAA the average elongation for a 24-hour period was about 3.5 mm as compared to 1.5-2.0 mm in wat’er. In general the results are the same as those shown in Fig. 1 except the stimulat’ing effect of ATP alone is not quite so marked. An enhancement of elongation of about 15 % resulted in ATP solutions of 1.25 and 2.5 X 1O-4 M, in IAA. Again 0.01 A// 1-ethionine inhibited elongation about 70%, and when ATP (2.5 X 1O-4 M) is added this is reduced to about 37%.
354
NORRIS
I
01
I 2
I
I 4
I 3
I 5
Cont.
of ATP
I 6
(X lO-4
I 7
I 8
I
I
5
10
Mob)
FIQ. 1. Influence of ATP on the elongation of the second 5-mm segment of the Avena coleoptile. (I) In a strongly inhibitory solution (0.01 M) of I-ethionine; (II) in water. Elongation is expressed as percentage of the water control. Standard deviations are shown. Each plotted point is the average of 1739 measurements. The figure represents the data of one typical experiment selected from a total of six replications. tions and partially reversing the ethionine inhibition of their elongation. This is true both in the presence and absence of exogenous IAA. From the preliminary
o
I
I I
I 2
I 3
Cont.
I 4
I 5
of ATP
(X d
I 6
I 7
I 8
Molar1
FIG. 2. Influence of ATP on the elongation of the second 5-mm segment of the Avena coleoptile
in IAA. (I) In a strongly inhibitory solution (0.01 M) of I-ethionine in IAA; (II) in IAA only. Elongation is expressed as percentage of the IAA control. Standard deviations are shown. Each plotted point is the average of 16-45 measurements. The figure represents the data of one typical experiment selected from a total of five replications. DISCUSSION It is evident from the data presented that the application of ATP is capable of stimulating the growth of Avena coleoptile sec-
work reported
here
no exact conclusions can be drawn as to the mechanisms involved. It may be recalled that Shull (8) noted that ethionine induced a marked decrease in the level of liver ATP, and in the later studies of Villa-Trevino et al. (7) this was observed to parallel the inhibition of protein synthesis. They also found that the administration of ATP or adenine counteracted both the inhibition of protein synthesis and the decreased level of hepatic ATP. These workers tested various compounds and found that only those convertible to adenine or an adenine derivative were effective, which led them to suggest that ethionine exerted its primary effect on the concentration of ATP and that the inhibition of protein synthesis was secondary. In an extensive discussion Villa-Trevino et al. (7) explored the possible manner in which ethionine affects the cellular level of ATP, and how adenine or ATP can counteract this effect, as well as the question as to how a decrease in ATP concentration inhibits the protein synthetic syst,em. They
REVERSAL
OF ETHIONINE
concluded that their data best support the idea detailed by Stekol et al. (9) and are supported by the work of Schmidt et al. (11) that ethionine exerts an adenine- or ATP-trapping effect. Briefly, based on Cantoni’s (12) original work, this involves the metabolic interaction of ATP with ethionine and methionine to form S-adenosylethionine and X-adenosylmethionine. Unlike the activated methionine, the X-adenosylethionine in effect represents a trap since the adenosine moiety is not readily released from this compound. With regard to the protein synthetic system, they suggest (7) that inhibition may be due to interference with the synthesis of “messenger RNA.” Webster (13) showed that ethionine blocks protein synthesis in plants, and both Schrank (5) and Cleland (6) suggested that the inhibitory effects of ethionine on elongation of the oat coleoptile might result from interference with protein synthesis. Nooden and Thimann (14) have recently presented evidence that some protein synthesis is required for auxin-induced cell enlargement. The above workers (5, 6, 13) did not specifically consider the manner in which inhibition of protein synthesis is brought about by ethionine, but the inference is on methionine-ethionine interaction. The data presented here are in agreement with the recently introduced concept that ethionine exerts its inhibitory effects by interference with ATP metabolism. The fact that ATP stimulates elongation of the oat coleoptile,
EFFECT
BY ATP
355
even in the absence of ethionine inhibition, might be taken as an indication that under normal conditions the metabolic energy supply (quantity of ATP) is not adequate to support maximal elongation. ACKNOWLEDGMENTS The technical assistance of Nick Vratis is acknowledged with pleasure. The author is indebted to Professor A. R. Schrank for criticism of the manuscript. REFERENCES 1. DYER, H. M., J. Biol. Chem. 134, 519 (1938). 2. SHAPIRO, S. K., AND SCHLENK, F., Advan. Enzymol. 22, 237 (1960). 3. FARBER, E., Advan. Cancer Res. 7, 383 (1963). 4. BOLL, W. G., Plant Phyeiol. 36, 115 (1960). 5. SCHRANK, A. R., Archiv. Biochem. Biophye. 61,
348 (1956). 6. CLELAND, It., Plant Physiol. 36, 5% (1960). 7. VILLA-TREVINO, S., SHULL, K. H., AND FARBER, E., J. Biol. Chem. 233, 1757 (1963). 8. SHULL, K. H., J. Biol. Chem. 237, PC1734 (1962). 9. STEKOL, J. A., ANDERSON, E. I., Hsu, P. T., AND WEISS, S., Abstr. 127th Meeting Am. Chem. SOL, Cincinnati, 1966~. 4C. American Chemical Society, Washington, D. C. 10. WIEGAND, 0. F., AND SCHRANK, A. R., Botan. Gaz. 121, 106 (1959). 11. SCHMIDT, G., SERAIDARIAN, K., GREENBAUM, L. M., HICKEY, M. D., AND THANNHAUSER, S. J., Biochim. Biophys. Acta 20, 135 (1956). 12. CANTONI, G. L., J. Biol. Chem. 189,745 (1951). 13. WEBSTER, G. C., Plant Physiol. 30, 351 (1955). 14. NOOD~N, L. D., AND THIMANN, K. V., Proc. Natl. Acad. Sci. U.S. 60, 194 (1963).