BIOINORGANIC CHEMSTR Y 6.229-232
Thallium
Antagonism
B. 2. SIEGEL
Toward
(1976)
Potassium
229
Dependent Systems
AND S. M. SIEGEL
Pacific Biomedical Research Center, and Departnzent of Botany, University of HpNaii, Horzolulu, Hawaii, 9682-3
ABSTRACT When a number of Group 1A chlorides were added to yeast solutions which were inhibited by 20 mM Tl +, the greatest restorative effect was shown by KCl, with a lesser effect by NH&l. Sodium, rubidium and cesium chlorides had no significant effect on Tlf-inhibited CO* production, and lithium chloride inhibited the system even further. Removal of potassium ions by dialysis reduced CO2 production by about 70% and the restorative effect of potassium was markedly reduced when this ion was added in the presence of thallium.
INTRODUCTION The clinical toxicity of thallium is well characterized [ l] , and I‘_._ so dangerous that neither unrestricted home uses nor field uses are permitted” [2]. In contrast, phytotoxicity of thallium has been the subject of only two reports [3,4]. Thallium may be of unusual ecological significance because it, like mercury, is a constituent of natural thermal emissions [S] _ We have recently shown that inhibition of Plantago seed germination and Aspergillrts mycelial growth by thallium (TIC) was reduced or reversed by the addition of potassium ions (K’), but not by sodium ions (Na*) [4] _ The phenomenon was explained on the basis of close size-charge relations between Tl+ and K’, but not Na+. It was further suggested that thallium toxicity couId be based upon a specific antagonism toward one or more of the K+-dependent enzymes or enzyme systems. Using K*dependent glycolytic COP production for we have now shown that inhibition by thallium can be largely reversed t319 by potassium, and to some extent by ammonium ions, but not by other members of group IA. Berrera and GomezPuyou [6] have additionally shown that the Tl+ inhibitory effect seems to be specific for K+ since it does not affect the movement of Na+ across the mitochondrial membrane_ Q American Elsevier Publishing Company, Inc., 1976
230
B. Z. SIEGEL AND S. M. SIEGEL MATERIALS AND METHODS
A iO% suspension of yeast (“Baker’s” Saccharomyces cereviceae) was made using distilled water and stored at 7 “C for not more than 3 days before use. Ahquots were first allowed to equilibrate with a 24 “C ambient air temperature before beginning each experiment _ For the measurement of CO2 production, 10 ml gas fermentation tubes graduated to 0.5 ml were used. For experimental trials, each tube contained a 1% yeast suspension, 3 @-D-d ucose with or without the salts of the ions to be tested. The experiments were run in quadruplicate and the rest&s recorded hourly for 4 hours. The pH of the solution mixtures was determined both at the beginning and termination of the experiments to be 5.7. This pH is nearly 0.7 units beIow the pK, of the CO-z-bicarbonate system, hence more than 80% of the COa evolved in the reaction was present as a gas and capable of measurement by displacement in the fermentation tubes_ Therefore, it was not deemed necessary to correct the data to absolute volumes of gas as the error is systematic_ Dialysis for IS-18 hours against distilled water at 10 “C was carried out to lower the endogenous Kf level of the yeast suspensions_ Dialyzed and undialyzed preparations from the same suspensions were always compared and all data presented were based upon at least triplicate preparations.
RESULTS AND DISCUSSION Twenty n&l TIC1 was used as the test concentration because it gave approximately 50% inhibition of the CO2 production, as compared with the control (no added Group 1A chlorides). The addition of Tl+ affects the rate of CO2 production but had no determinable effect on the onset of gas production, Le., did not produce a lag phase_ When a number of Group 1 A (40 niM) chlorides were added to yeast solutions which were inhibited by 20 mM Tl+ the greatest restorative effect was shown by KCl, with a lesser effect produced by NH4C1 (Table 1). Sodium. rubidium and cesium chlorides had no significant effect on Tl’inhibited CO2 production; LiCl appeared to inhibit the system even further_ In contrast, the addition of 40 mM concentrations of group IA salts to yeast suspensions in the absence of thallium, in no case exhibited a significant effect upon CO2 evolution_ The effectiveness of dialysis (Table 2) in removing potassium ions is reflected in the drop by about 70% in CO, production, and the restoration of a large part of this differential upon addition of K+_ The selectivity of the restoration process is shown in the failure of sodium ions to replace potassium ions. The fact
Tl+ ANTAGONISM
TOWARD
K” DEPENDENT
TABLE
SYSTEMS
231
1
The Relative Effectiveness of KC1 and Other Group 1A Chlorides Against Thallium-Inhibited Yeast Glycolysis Radius, M+ Ionic additions
Control 20 mM TIC1 20 mM TIC1 + 40 mM LiCl 20 mM TICI + 40 mM NaCl 20 mM TlCl + 40 mM KC1 20 mM TIC1 + 20 mM NH&Cl 20 mM TlCl + 40 mM RbCl 20 mM TIC1 + 40 mM CsCl
@>
8.3 3.7 2.8 3.3 6.0 5.0 3.9 4.1
1.47 0.68 0.97 1.33 1.43 1.47 I.67
TABLE
COa produced cm314 hr
20.4 + 0.2 + 0.1 + 0.2 * 0.3 f 0.2 + 0.2 2 0.2
COa produced of control 100
45 33 40 72 60 47 50
2
Sensitivity of Glycolysis to Thallium and Potassium Chlorides in Dialyzed and Undialyzed Yeast Preparations Relative Cog
production
TlCl
Salt added KC1
NaCl
CrnM)
(mM)
(mM)
Non-diaIyzed
Diaiyzed
0 20 0 0 20
0 0 40 0 40
0 0 0 40 0
100 43 93 90 71
29 21 80 25 61
that glycolysis was incompletely restored reflects upon the removal in part of other cofactors such as metal ions and nucleotides which were, of course, not replaced_ In this system the low baseline production of CO* is not further diminished significantly by the addition of thallium. But the restorative effect of potassium was markedly reduced when this ion was added in the presence of thallium. These d&a extended further the previous evidence for a specific K+ - Tl+ antagonism. They also pose problems for further investigation: what ionic properties account for the observed differences between Rb+ and NH,* and what is the nature of Tl*-insensitive COa production by dialyzed preparations.
B. Z. SIEGEL ANJ3 S. M. SIEGEL
232
REFERENCES 1. N. Kanrerbeek, Therapeutic probIems in thallium poisoning,
Dissertation, Dr. Med., Rijjuniversiteit of Utrecht. the Netherlands (1971). 2_ Special communication. C&z_ ToxicoL 5,89 (1972). 3. Harold 3. Evans and George J. Sorger, Ann. Rev- of Phr Physiol. 17,45 (1966)4_ B_ Z_ Siegel and S_ M. Siegel. Biotiorg. Chem 4,93 (197.5). 5. G. SigvaIdason, VolcanoIogical Institute, University of Iceland, Reykjavik, Iceland, personal communication (1973). 6.
Hector
Barrera and A. Gomez-Puyou,
Received 8 March I9 76.
J. Biol. Chem 250,537O
(1974).
Thallium
Antagonism
B. 2. SIEGEL
Toward
(1976)
Potassium
229
Dependent Systems
AND S. M. SIEGEL
Pacific Biomedical Research Center, and Departnzent of Botany, University of HpNaii, Horzolulu, Hawaii, 9682-3
ABSTRACT When a number of Group 1A chlorides were added to yeast solutions which were inhibited by 20 mM Tl +, the greatest restorative effect was shown by KCl, with a lesser effect by NH&l. Sodium, rubidium and cesium chlorides had no significant effect on Tlf-inhibited CO* production, and lithium chloride inhibited the system even further. Removal of potassium ions by dialysis reduced CO2 production by about 70% and the restorative effect of potassium was markedly reduced when this ion was added in the presence of thallium.
INTRODUCTION The clinical toxicity of thallium is well characterized [ l] , and I‘_._ so dangerous that neither unrestricted home uses nor field uses are permitted” [2]. In contrast, phytotoxicity of thallium has been the subject of only two reports [3,4]. Thallium may be of unusual ecological significance because it, like mercury, is a constituent of natural thermal emissions [S] _ We have recently shown that inhibition of Plantago seed germination and Aspergillrts mycelial growth by thallium (TIC) was reduced or reversed by the addition of potassium ions (K’), but not by sodium ions (Na*) [4] _ The phenomenon was explained on the basis of close size-charge relations between Tl+ and K’, but not Na+. It was further suggested that thallium toxicity couId be based upon a specific antagonism toward one or more of the K+-dependent enzymes or enzyme systems. Using K*dependent glycolytic COP production for we have now shown that inhibition by thallium can be largely reversed t319 by potassium, and to some extent by ammonium ions, but not by other members of group IA. Berrera and GomezPuyou [6] have additionally shown that the Tl+ inhibitory effect seems to be specific for K+ since it does not affect the movement of Na+ across the mitochondrial membrane_ Q American Elsevier Publishing Company, Inc., 1976
230
B. Z. SIEGEL AND S. M. SIEGEL MATERIALS AND METHODS
A iO% suspension of yeast (“Baker’s” Saccharomyces cereviceae) was made using distilled water and stored at 7 “C for not more than 3 days before use. Ahquots were first allowed to equilibrate with a 24 “C ambient air temperature before beginning each experiment _ For the measurement of CO2 production, 10 ml gas fermentation tubes graduated to 0.5 ml were used. For experimental trials, each tube contained a 1% yeast suspension, 3 @-D-d ucose with or without the salts of the ions to be tested. The experiments were run in quadruplicate and the rest&s recorded hourly for 4 hours. The pH of the solution mixtures was determined both at the beginning and termination of the experiments to be 5.7. This pH is nearly 0.7 units beIow the pK, of the CO-z-bicarbonate system, hence more than 80% of the COa evolved in the reaction was present as a gas and capable of measurement by displacement in the fermentation tubes_ Therefore, it was not deemed necessary to correct the data to absolute volumes of gas as the error is systematic_ Dialysis for IS-18 hours against distilled water at 10 “C was carried out to lower the endogenous Kf level of the yeast suspensions_ Dialyzed and undialyzed preparations from the same suspensions were always compared and all data presented were based upon at least triplicate preparations.
RESULTS AND DISCUSSION Twenty n&l TIC1 was used as the test concentration because it gave approximately 50% inhibition of the CO2 production, as compared with the control (no added Group 1A chlorides). The addition of Tl+ affects the rate of CO2 production but had no determinable effect on the onset of gas production, Le., did not produce a lag phase_ When a number of Group 1 A (40 niM) chlorides were added to yeast solutions which were inhibited by 20 mM Tl+ the greatest restorative effect was shown by KCl, with a lesser effect produced by NH4C1 (Table 1). Sodium. rubidium and cesium chlorides had no significant effect on Tl’inhibited CO2 production; LiCl appeared to inhibit the system even further_ In contrast, the addition of 40 mM concentrations of group IA salts to yeast suspensions in the absence of thallium, in no case exhibited a significant effect upon CO2 evolution_ The effectiveness of dialysis (Table 2) in removing potassium ions is reflected in the drop by about 70% in CO, production, and the restoration of a large part of this differential upon addition of K+_ The selectivity of the restoration process is shown in the failure of sodium ions to replace potassium ions. The fact
Tl+ ANTAGONISM
TOWARD
K” DEPENDENT
TABLE
SYSTEMS
231
1
The Relative Effectiveness of KC1 and Other Group 1A Chlorides Against Thallium-Inhibited Yeast Glycolysis Radius, M+ Ionic additions
Control 20 mM TIC1 20 mM TIC1 + 40 mM LiCl 20 mM TICI + 40 mM NaCl 20 mM TlCl + 40 mM KC1 20 mM TIC1 + 20 mM NH&Cl 20 mM TlCl + 40 mM RbCl 20 mM TIC1 + 40 mM CsCl
@>
8.3 3.7 2.8 3.3 6.0 5.0 3.9 4.1
1.47 0.68 0.97 1.33 1.43 1.47 I.67
TABLE
COa produced cm314 hr
20.4 + 0.2 + 0.1 + 0.2 * 0.3 f 0.2 + 0.2 2 0.2
COa produced of control 100
45 33 40 72 60 47 50
2
Sensitivity of Glycolysis to Thallium and Potassium Chlorides in Dialyzed and Undialyzed Yeast Preparations Relative Cog
production
TlCl
Salt added KC1
NaCl
CrnM)
(mM)
(mM)
Non-diaIyzed
Diaiyzed
0 20 0 0 20
0 0 40 0 40
0 0 0 40 0
100 43 93 90 71
29 21 80 25 61
that glycolysis was incompletely restored reflects upon the removal in part of other cofactors such as metal ions and nucleotides which were, of course, not replaced_ In this system the low baseline production of CO* is not further diminished significantly by the addition of thallium. But the restorative effect of potassium was markedly reduced when this ion was added in the presence of thallium. These d&a extended further the previous evidence for a specific K+ - Tl+ antagonism. They also pose problems for further investigation: what ionic properties account for the observed differences between Rb+ and NH,* and what is the nature of Tl*-insensitive COa production by dialyzed preparations.
B. Z. SIEGEL ANJ3 S. M. SIEGEL
232
REFERENCES 1. N. Kanrerbeek, Therapeutic probIems in thallium poisoning,
Dissertation, Dr. Med., Rijjuniversiteit of Utrecht. the Netherlands (1971). 2_ Special communication. C&z_ ToxicoL 5,89 (1972). 3. Harold 3. Evans and George J. Sorger, Ann. Rev- of Phr Physiol. 17,45 (1966)4_ B_ Z_ Siegel and S_ M. Siegel. Biotiorg. Chem 4,93 (197.5). 5. G. SigvaIdason, VolcanoIogical Institute, University of Iceland, Reykjavik, Iceland, personal communication (1973). 6.
Hector
Barrera and A. Gomez-Puyou,
Received 8 March I9 76.
J. Biol. Chem 250,537O
(1974).
