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Tracer experiments on the formation of ethylene by apple tissue
LETTERS
Tracer
Experiments
TO
THE
on the Formation
543
EDITORS
of Ethylene
by Apple Tissue’
The production of ethylene by a variety of plant materials, and the acceleratory influence which the gas exerts on the process of fruit ripening, are well substantiated facts. However, the metabolic pathway giving rise to ethylene is only partially understood, and it is likely that experiments involving labeled substrates will be essential for the complete elucidation of the process. This communication outlines a simple procedure for determining C2”H, , and describes some results which have been obtained when labeled substrates were soaked into apple sections. Plugs of tissue cut from McIntosh apples were placed in a radioactive solution and after 60 min. they were rinsed, dried, and inserted in a narrow tube having a glass fitting in the middle and stopcocks at both ends. The tube was constructed to hold exactly the amount of tissue employed (ca. 40 g.). A 500 ml. Florence flask containing 2 ml. of mercuric perchlorate solution (6) was sealed with a glass fitting having a stopcock, and then attached by rubber tubing to one end of the tube containing the tissue. For sampling the gas, the stopcocks between the flask and the tube were opened, and when the air had equilibrated between the two vessels, the stopcock at the further end of the tube was opened to flush the remaining gas into the flask. At this time the tissue was either killed in boiling 50% ethanol, or a second collection period was initiated. Absorption of ethylene was accomplished by rotating the Florence flask (at 4°C.) on a special shaker, and was found to be complete after 30 min. The apparatus shown in Fig. 1 was used to release the ethylene from the absorbent, the gas being collected over water in a vaccine bottle. The solubility of ethylene in water is so low that no detectable loss occurs under the conditions of the release, and quantities as small as 10-Z pl. have been quantitatively recovered by this method. The entire gaseous sample (ca. 0.2 ml.) was then withdrawn from the vaccine bottle with a 1 ml. syringe. Possible traces of carbon dioxide were removed by drawing 0.3 ml. of 1 N NaOH into the syringe and rolling the air bubble back and forth through the solution. All but a few drops of NaOH were then forced out, and the gas sample was adjusted to exactly 0.75 ml. volume by drawing air into the syringe. This final volume was replaced over water in the vaccine bottle. A dr,y 1 ml. syringe was used to remove 0.5 ml. of the sample and inject it through a rubber fitting into an evacuated Ballentine counting tube. The remaining 0.25 ml. sample was gas chromatographed to determine the amount of ethylene present (2,3). The Ballentine tube was filled with 10% methane-90Y0 argon mixture to one atmosphere pressure and radioactivity was determined. The counting characteristics of the tube were not affected by 0.5 ml. of water-saturated air. In this procedure, the identification of ethylene is based on a number of properties: only olefins are absorbed on Hg(Cl0 4) 2 and released from the complex by LiCl (6); the released gas chromatographs on aluminum oxide with the same retention time as synthetic ethylene but not other olefins (2, 3); ethylene has low solubility in water and alkali; ethylene is known to be produced by apples and in amounts comparable with those found in these experiments (3, 4) ; in addition (see procedure in Table I, footnote a) ethylene is the only olefin volatile at -78°C. and condensed at - 196°C. When a 57, solution of 1,3-Cl4 glycerol2 (S.A., 256 dpm/pg. C) was fed to apple sections, no activity appeared in the total ethylene accumulated during 7 hr. Analysis of a tissue extract made at that time showed significant activity in glutamate (4.7), 1 This work was supported by a grant to Prof. K. V. Thimann, Harvard University, from the Maria Moors Cabot Foundation. 2 Kindly supplied by Dr. Manfred L. Karnovsky, Harvard Medical School.
544
LETTERS
TO
THE
EDITORS
for releasing ethylene from mercuric perchlorate. With 2 m FIG. 1. Apparatus of 4 N LiCl in E and stopcocks A and B in position 3, the unit is evacuated with a vacuum pump through G. Stopcock A is then turned to position 2 and the vacuum pump disconnected. The mercuric perchlorate sampleis drawn into tube E from beaker D by opening stopcock C slightly. Tube E is immersed in a waterbath (50°C.) for 3 min., which causes the mixture of mercuric perchlorate and LiCl to reflux. After removing the water bath, hot water is introduced into tube E, through C, until the level of water rises above stopcock B. Stopcock B is turned to position 1, allowing water to enter tube F from the reservoir. Stopcock A is turned to position 1, causing water from the reservoir to flush out tube G and overflow into the bulb. A bottomless vaccine bottle is placed over tube G so that its lower lip is immersed in the water contained in the bulb, and the air in the vaccine bottle is withdrawn by means of a hypodermic syringe inserted through the vaccine cap. When stopcock A is turned to position 3, the air sample containing ethylene rises from tube F through G to collect in the vaccine bottle.
LETTERS
TO
THE
TABLE Production
545
EDITORS
I
of V402 and Cg14Hdfrom Cl4 Evenly CH&‘400Na
(Substrate:” 100.2 mg. of sucrose (0.75 pc./mg.), acetate (12.1 fic./mg.), 1 experimentc.)
Labeled
2 experiments;b
Total
Sucrose and 8.2 mg. of sodium
cts./min.
(t22 hr.
23-58 hr.
ethylene
carbon dioxide
ethylene
carbon dioxide
Sucrose Sucrose Acetate
(I) (II)
1052 300 551
560,000 398,000 10,200,000
11,285 2,440 774
5,360,OOO 3,580,OOO 40,500,000
Sucrose Sucrose Acetate
(I) (II)
5400 3500 4140
21,100 21,100 432,000
Cts./min./mg.
carbon 24,150 5,950 1,880
89,000 75,700 762,500
u The tracer was dissolved in 2 ml. of water and injected into the ovary of an apple. Air was passed over the fruit and carbon dioxide collected with a NaOH column, and ethylene with a mercuric perchlorate column. Ethylene was released from the absorbent, by the addition of LiCl, and then distilled through a dry ice trap into an evacuated counting tube maintained in liquid nitrogen. b Kindly supplied by Prof. G. Krotkov, Queen’s University. c Obtained from Prof. K. Bloch, Harvard University. aspartate (3.6), malate (4.0), glucose (0.3), fructose (3.9), and sucrose (6.3), and higher specific activities in alanine (211) and succinate (23) (all figures are dpm/hg. C). In a shorter experiment these intermediates were found to have reached very nearly the same specific activity 15 min. after the feeding was completed. A similar experiment was carried out with 2-V glycerol2 and again no label could be detected in the ethylene collected during a 7 hr. period. In each of the 7 hr. experiments ca. 15pg. of ethylene was produced, and since the counting procedure can reliably detect about 10 cts./min. at 90% efficiency, the specific activity of the ethylene could not have been as high as 1 dpm/pg. carbon. In 1957, Phan-Ch6n-Ton found that Penicililum digitatum produced far more ethylene when it was grown on a medium containing 2% glycerol than with 2% sucrose as a carbon source, and he tentatively concluded that a 3-carbon compound metabolically related to glycerol might be a precursor of ethylene (5). Burg and Thimann (4) found that high glycerol concentrations maintained the ethylene production of apple sections, whereas soaking in water caused an inhibition of ethylene production. However, from other evidence (for instance, the fact that KC1 exerted a similar influence) the authors concluded that the effect was osmotic in nature. Although no label appeared in ethylene after C’4glycerol feedings, the tissue did show its characteristic response to the substance, and the glycerol-treated tissue produced ethylene at 25Oojn the rate of water-soaked controls. At least in the apple, therefore, glycerol is not a substrate for ethylene production and the enhancement of ethylene production observed in its presence must be due to an osmotic response. In experiments with whole apples, using somewhat different counting and collecting procedures, it was found that evenly labeled Cl4 sucrose injected into the ovary
546
LETTERS
TO
THE
EDITORS
of the fruit did give rise to small amounts of labeled ethylene, whereas Cl4 carboxyllabeled acetate was a less effective precursor (Table I). Although these experiments were of relatively long duration, they do demonstrate the incorporation of label into ethylene. However, especially with acetate, so little incorporation occurred relat,ive to that in carbon dioxide that it may be concluded that ethylene production is not associated with Krebs cycle organic acid metabolism. The specific activity of succinate after Cl4 glycerol feeding (see above) tends to support this conclusion, while the high activity of alanine suggests that glycolysis intermediates are also not direct precursors of ethylene. Since Cl4 ethylene has been shown in ripe pears and avocados to transfer activity to fumaric and succinic acids, and to a lesser extent to sugar (l), it seemed possible that ethylene might be derived from aromatic compounds originating from sugar, and that after incorporation of labeled ethylene the label appeared in degradation products of the aromatic compounds. This was investigated by feeding evenly labeled Cl4 tyrosine (S.A., 0.74 pc./mg.) in 2% KC1 solution to apple sections. Although at least 5y0 of the activity was recovered as Cl402 , indicating that tyrosine had been metabolized, no radioactivity could be detected in ethylene even after 12 hr. It seems, therefore, that the precursors of ethylene may be quite distinct from major pathways of metabolism; a fact which is consistent with the very small quantities of the gas which are normally produced in biological systems. REFERENCES 1. BUHLER, D. R., HANSEN, E., AND WANG, 2. BURG, S. P., AND STOLWIJK, J. A. J., J. (in press). 3. BURG, S. P., AND THIMANN, K. V., Proc. 4. BURG, S. P., AND THIMANN, K. V., Plant 5. PHAN-C&N-TON, Compt. rend. acad. sci. 6. YOUNG, R. E., PRATT, H. K., AND BIALE,
C. H., Nature 1’79, 48 (1957). B&hem. and Microbial. Tech. and Eng. Natl. Acad. Sci. 46, 335 (1959). Physiol. (in press). 244, 1243 (1957). J. B., Am. J. Botany 36, 814 (1948). STANLEY P. BURGS
Biological Department Harvard University Cambridge, Massachusetts Received May 9, 1969
Effect
of Valinomycin
on Oxidative
Phosphorylation
Inhibitory effects of several toxic antibiotics on oxidation and phosphorylation reactions in mammalian liver mitochondria have been reported recently by Lardy et al. (1). Our studies demonstrate that the antibiotic valinomycin (2), produces uncoupling of oxidative phosphorylation and activation of ATPase in rat liver mitochondria at very low concentrations. Mitochondria were prepared from rat liver as described by Schneider (3), and assayed for oxidative phosphorylation efficiency according to the method of Maley and Lardy (4). 3 This investigation was carried out while the author was fellowship from the National Cancer Institute, U. S. Public G. Krotkov, Queen’s University, Dr. Bruce B. Stowe, Harvard W. Samuel, Harvard University, and Prof. K. V. Thimann, provided invaluable suggestions and assistance.
holder of a research Health Service. Prof. University, Edmund Harvard University,
Tracer
Experiments
TO
THE
on the Formation
543
EDITORS
of Ethylene
by Apple Tissue’
The production of ethylene by a variety of plant materials, and the acceleratory influence which the gas exerts on the process of fruit ripening, are well substantiated facts. However, the metabolic pathway giving rise to ethylene is only partially understood, and it is likely that experiments involving labeled substrates will be essential for the complete elucidation of the process. This communication outlines a simple procedure for determining C2”H, , and describes some results which have been obtained when labeled substrates were soaked into apple sections. Plugs of tissue cut from McIntosh apples were placed in a radioactive solution and after 60 min. they were rinsed, dried, and inserted in a narrow tube having a glass fitting in the middle and stopcocks at both ends. The tube was constructed to hold exactly the amount of tissue employed (ca. 40 g.). A 500 ml. Florence flask containing 2 ml. of mercuric perchlorate solution (6) was sealed with a glass fitting having a stopcock, and then attached by rubber tubing to one end of the tube containing the tissue. For sampling the gas, the stopcocks between the flask and the tube were opened, and when the air had equilibrated between the two vessels, the stopcock at the further end of the tube was opened to flush the remaining gas into the flask. At this time the tissue was either killed in boiling 50% ethanol, or a second collection period was initiated. Absorption of ethylene was accomplished by rotating the Florence flask (at 4°C.) on a special shaker, and was found to be complete after 30 min. The apparatus shown in Fig. 1 was used to release the ethylene from the absorbent, the gas being collected over water in a vaccine bottle. The solubility of ethylene in water is so low that no detectable loss occurs under the conditions of the release, and quantities as small as 10-Z pl. have been quantitatively recovered by this method. The entire gaseous sample (ca. 0.2 ml.) was then withdrawn from the vaccine bottle with a 1 ml. syringe. Possible traces of carbon dioxide were removed by drawing 0.3 ml. of 1 N NaOH into the syringe and rolling the air bubble back and forth through the solution. All but a few drops of NaOH were then forced out, and the gas sample was adjusted to exactly 0.75 ml. volume by drawing air into the syringe. This final volume was replaced over water in the vaccine bottle. A dr,y 1 ml. syringe was used to remove 0.5 ml. of the sample and inject it through a rubber fitting into an evacuated Ballentine counting tube. The remaining 0.25 ml. sample was gas chromatographed to determine the amount of ethylene present (2,3). The Ballentine tube was filled with 10% methane-90Y0 argon mixture to one atmosphere pressure and radioactivity was determined. The counting characteristics of the tube were not affected by 0.5 ml. of water-saturated air. In this procedure, the identification of ethylene is based on a number of properties: only olefins are absorbed on Hg(Cl0 4) 2 and released from the complex by LiCl (6); the released gas chromatographs on aluminum oxide with the same retention time as synthetic ethylene but not other olefins (2, 3); ethylene has low solubility in water and alkali; ethylene is known to be produced by apples and in amounts comparable with those found in these experiments (3, 4) ; in addition (see procedure in Table I, footnote a) ethylene is the only olefin volatile at -78°C. and condensed at - 196°C. When a 57, solution of 1,3-Cl4 glycerol2 (S.A., 256 dpm/pg. C) was fed to apple sections, no activity appeared in the total ethylene accumulated during 7 hr. Analysis of a tissue extract made at that time showed significant activity in glutamate (4.7), 1 This work was supported by a grant to Prof. K. V. Thimann, Harvard University, from the Maria Moors Cabot Foundation. 2 Kindly supplied by Dr. Manfred L. Karnovsky, Harvard Medical School.
544
LETTERS
TO
THE
EDITORS
for releasing ethylene from mercuric perchlorate. With 2 m FIG. 1. Apparatus of 4 N LiCl in E and stopcocks A and B in position 3, the unit is evacuated with a vacuum pump through G. Stopcock A is then turned to position 2 and the vacuum pump disconnected. The mercuric perchlorate sampleis drawn into tube E from beaker D by opening stopcock C slightly. Tube E is immersed in a waterbath (50°C.) for 3 min., which causes the mixture of mercuric perchlorate and LiCl to reflux. After removing the water bath, hot water is introduced into tube E, through C, until the level of water rises above stopcock B. Stopcock B is turned to position 1, allowing water to enter tube F from the reservoir. Stopcock A is turned to position 1, causing water from the reservoir to flush out tube G and overflow into the bulb. A bottomless vaccine bottle is placed over tube G so that its lower lip is immersed in the water contained in the bulb, and the air in the vaccine bottle is withdrawn by means of a hypodermic syringe inserted through the vaccine cap. When stopcock A is turned to position 3, the air sample containing ethylene rises from tube F through G to collect in the vaccine bottle.
LETTERS
TO
THE
TABLE Production
545
EDITORS
I
of V402 and Cg14Hdfrom Cl4 Evenly CH&‘400Na
(Substrate:” 100.2 mg. of sucrose (0.75 pc./mg.), acetate (12.1 fic./mg.), 1 experimentc.)
Labeled
2 experiments;b
Total
Sucrose and 8.2 mg. of sodium
cts./min.
(t22 hr.
23-58 hr.
ethylene
carbon dioxide
ethylene
carbon dioxide
Sucrose Sucrose Acetate
(I) (II)
1052 300 551
560,000 398,000 10,200,000
11,285 2,440 774
5,360,OOO 3,580,OOO 40,500,000
Sucrose Sucrose Acetate
(I) (II)
5400 3500 4140
21,100 21,100 432,000
Cts./min./mg.
carbon 24,150 5,950 1,880
89,000 75,700 762,500
u The tracer was dissolved in 2 ml. of water and injected into the ovary of an apple. Air was passed over the fruit and carbon dioxide collected with a NaOH column, and ethylene with a mercuric perchlorate column. Ethylene was released from the absorbent, by the addition of LiCl, and then distilled through a dry ice trap into an evacuated counting tube maintained in liquid nitrogen. b Kindly supplied by Prof. G. Krotkov, Queen’s University. c Obtained from Prof. K. Bloch, Harvard University. aspartate (3.6), malate (4.0), glucose (0.3), fructose (3.9), and sucrose (6.3), and higher specific activities in alanine (211) and succinate (23) (all figures are dpm/hg. C). In a shorter experiment these intermediates were found to have reached very nearly the same specific activity 15 min. after the feeding was completed. A similar experiment was carried out with 2-V glycerol2 and again no label could be detected in the ethylene collected during a 7 hr. period. In each of the 7 hr. experiments ca. 15pg. of ethylene was produced, and since the counting procedure can reliably detect about 10 cts./min. at 90% efficiency, the specific activity of the ethylene could not have been as high as 1 dpm/pg. carbon. In 1957, Phan-Ch6n-Ton found that Penicililum digitatum produced far more ethylene when it was grown on a medium containing 2% glycerol than with 2% sucrose as a carbon source, and he tentatively concluded that a 3-carbon compound metabolically related to glycerol might be a precursor of ethylene (5). Burg and Thimann (4) found that high glycerol concentrations maintained the ethylene production of apple sections, whereas soaking in water caused an inhibition of ethylene production. However, from other evidence (for instance, the fact that KC1 exerted a similar influence) the authors concluded that the effect was osmotic in nature. Although no label appeared in ethylene after C’4glycerol feedings, the tissue did show its characteristic response to the substance, and the glycerol-treated tissue produced ethylene at 25Oojn the rate of water-soaked controls. At least in the apple, therefore, glycerol is not a substrate for ethylene production and the enhancement of ethylene production observed in its presence must be due to an osmotic response. In experiments with whole apples, using somewhat different counting and collecting procedures, it was found that evenly labeled Cl4 sucrose injected into the ovary
546
LETTERS
TO
THE
EDITORS
of the fruit did give rise to small amounts of labeled ethylene, whereas Cl4 carboxyllabeled acetate was a less effective precursor (Table I). Although these experiments were of relatively long duration, they do demonstrate the incorporation of label into ethylene. However, especially with acetate, so little incorporation occurred relat,ive to that in carbon dioxide that it may be concluded that ethylene production is not associated with Krebs cycle organic acid metabolism. The specific activity of succinate after Cl4 glycerol feeding (see above) tends to support this conclusion, while the high activity of alanine suggests that glycolysis intermediates are also not direct precursors of ethylene. Since Cl4 ethylene has been shown in ripe pears and avocados to transfer activity to fumaric and succinic acids, and to a lesser extent to sugar (l), it seemed possible that ethylene might be derived from aromatic compounds originating from sugar, and that after incorporation of labeled ethylene the label appeared in degradation products of the aromatic compounds. This was investigated by feeding evenly labeled Cl4 tyrosine (S.A., 0.74 pc./mg.) in 2% KC1 solution to apple sections. Although at least 5y0 of the activity was recovered as Cl402 , indicating that tyrosine had been metabolized, no radioactivity could be detected in ethylene even after 12 hr. It seems, therefore, that the precursors of ethylene may be quite distinct from major pathways of metabolism; a fact which is consistent with the very small quantities of the gas which are normally produced in biological systems. REFERENCES 1. BUHLER, D. R., HANSEN, E., AND WANG, 2. BURG, S. P., AND STOLWIJK, J. A. J., J. (in press). 3. BURG, S. P., AND THIMANN, K. V., Proc. 4. BURG, S. P., AND THIMANN, K. V., Plant 5. PHAN-C&N-TON, Compt. rend. acad. sci. 6. YOUNG, R. E., PRATT, H. K., AND BIALE,
C. H., Nature 1’79, 48 (1957). B&hem. and Microbial. Tech. and Eng. Natl. Acad. Sci. 46, 335 (1959). Physiol. (in press). 244, 1243 (1957). J. B., Am. J. Botany 36, 814 (1948). STANLEY P. BURGS
Biological Department Harvard University Cambridge, Massachusetts Received May 9, 1969
Effect
of Valinomycin
on Oxidative
Phosphorylation
Inhibitory effects of several toxic antibiotics on oxidation and phosphorylation reactions in mammalian liver mitochondria have been reported recently by Lardy et al. (1). Our studies demonstrate that the antibiotic valinomycin (2), produces uncoupling of oxidative phosphorylation and activation of ATPase in rat liver mitochondria at very low concentrations. Mitochondria were prepared from rat liver as described by Schneider (3), and assayed for oxidative phosphorylation efficiency according to the method of Maley and Lardy (4). 3 This investigation was carried out while the author was fellowship from the National Cancer Institute, U. S. Public G. Krotkov, Queen’s University, Dr. Bruce B. Stowe, Harvard W. Samuel, Harvard University, and Prof. K. V. Thimann, provided invaluable suggestions and assistance.
holder of a research Health Service. Prof. University, Edmund Harvard University,