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The reversible depolymerization of actin by potassium iodide
The Reversible Depolymerization by Potassium Iodide Andrew FT~
the Institute
G. Szent-GySrgyi
GOT Muscle Research at the Marine Woods Hole, Massachusetts Received
November
of Actin 1 Biological
Laboratory,
14, 1950
Actin prepared according to the method of Straub (1) is characterized by its ability to exist in two forms. In salt-free condition it is present in the globular form (G-actin); in the presence of salts it polymerizes into long fibers (F-actin). As shown by Rozsa, Szent-Gyorgyi and Wyckoff (2), this polymerization occurs in two steps: first a number of molecules form larger globules, then these globules unite to form fibers. Straub (1) showed that 0.6 M potassium iodide depolymerizes F-actin. Guba (3) found this effect to be irreversible and instantaneous. In the present paper it is shown that the KI-depolymerization can take place reversibly: data are presented on the changes in the adenine nucleotide content of actin during polymerization and depolymerization. METHODS Actin, adenosine triphosphate (ATP), inosine triphosphate (ITP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were prepared as described previously (4). A stock solution of 6.0 M KI was prepared containing 0.06 M sodium thiosulfate, which prevented the formation of free iodide. The solution was kept in a dark bottle. For the determination of ATP, ADP, and AMP, the enzymatic, differential spectrophotometric method of Kalckar (5) was used, following his description closely. Myokinase (6), desamidase (7), and potato apyrase (7) were prepared as described by Kalckar. The spectrophotometric measurements were made in a Beckman model D. U. spectrophotometer. For the determination of the nucleotides, 4 ml. of the actin solutions was placed in boiling water for 5 min. after each experiment. Then 1 drop of 2 M pH 4.7 sodium acetate buffer was added to the warm actin solutions, which were then filtered, the protein-free liquids being used for the determination. One ml. 1 This
research
was supported
by a grant 97
from
the American
Heart
Association.
98
ANDREW
0. SZENT-QYijRGYI
of the filtrate was diluted with 0.5 ml. water and 1.5 ml. of pH 6.1 0.33 M succinate buffer. The extinction was measured according to Kalckar, his factor being used. The experiments and measurements were made at room temperature, except where otherwise stated. The experiments on G-actin were performed immediately after actin was extracted from the acetone-dried muscle powder. RESULTS
AND DISCUSSION
The E$ect of KI on Actin G-actin, as obtained directly from the acetone-dried muscle powder, polymerizes at almost the same rate and to the same level in 0.1 M KI as in KCl, judging by its viscosity. If this G-a&in is incubated in 0.6 M
83
2
I
I Min.
0
IO
20
30
Fro. 1. Polymerization of G-&in with Polymerization of G-a&in in 0.1 M KCI. 0.1 M KI after incubation in 0.6 M KI for contained 0.0016 M MgClz and 0.0067 M
40
I 50
60
70
and without KI treatment. Empty circles: Semicircles: Polymerization of G-actin in 20 min. at room temperature. Both samples pH 7.0 phosphate buffer.
KI 10 or 20 min. at room temperature and then diluted to contain 0.1 44 KI, the polymerization is still fairly quick and complete, as shown in Fig. 1. If, however, F-actin is subjected to the same treatment, no polymerization takes place. Ten minutes’ incubation of actin in its F-form in 0.6 M KI is more than enough to cause complete and irreversible depolymerization. F-actin incubated with 0.6 M KI for a few minutes depolymerizes completely and will not polymerize again after diluting it to 0.1 M KI concentration. This shows that there is a differ-
ACTIN
99
DEPOLYMERIZATJON
ence between the two G-actins, one obtained directly from the acetonedried muscle powder, the other from F-actin via depolymerization, the latter being extremely labile. The depolymerization of F-actin by KI can be reversed if ATP is added before depolymerization takes place. In this case the G-actin obtained will polymerize again and show the original viscosity of F-actin in about 30 min. The presence of ATP is needed during depolymeriza-
Min.
0
IO
20
30
Fro. 2. Repolymerization of actin. F-a&in was treated with 0.6 M KI for 10 min. and diluted to 0.1 M KI (O-time) in the presence of 0.0016 M MgCl* and 0.0667 A4 pH 7.0 phosphate bufIer. Empty circles: lo+ mole ATP added immediately before addition of 0.6 M KI (ATP concentration calculated for the volume before dilution). Semicircles: 10-s mole ATP added immediately before dilution. Circles with dots: no ATP added. vaDof original F-actin: 0.38.
tion if this process is to be reversible. Adenosine triphosphate added after depolymerization is ineffective (Fig. 2.). The described effect of ATP on actin depolymerization is in accord with the findings of Straub and Feuer (8), according to which depolymerization of F-actin by dialysis against distilled water is reversible only in the presence of ATP. It is also in agreement with the experiments described in the foregoing paper (4), in which depolymerization by urea was found reversible only in the presence of ATP. In both cases ATP was effective only if added before depolymerization. While the
100
ANDREW
G.
SZENT-GYijRGYI
reversible depolymerization by dialysis takes days, and the reversible depolymerization by urea takes hours, the reversible depolymerization by KI can be effected within minutes. This action of ATP was not specific as in the case of urea and contrary to the dialysis experiments of Straub and Feuer (8). Both ADP and ITP were effective in the same amounts as ATP, the lower limit of maximal effect with all three nucleotides lying around 5 X 10m6mole
Fra. 3. Protective action of ATP (vertical semicircles), ADP (black and white semicircies, white top), ITP (black and white semicircles, black top) and AMP (empty circles). Actin was treated for 10 min. with 0.6 M KI in the presence of the nucleotides of diffeferent concentrations (abscissa), added immediately before treatment with KI. Then the solution was diluted to contain 0.1 M KI in the presence of 0.0016 M MgClz and 0.0067 M pH 7.0 phosphate buffer and the viscosity read an hour later (ordinate). qsp of original F-a&in: 0.38.
(Fig. 3). Actin, prepared by Straub’s method, contains myokinase and desamidase, as shown by Laki and Clark (9), making difficult the interpretation of the experiments with ADP. But the fact that ADP was active in as low concentrations as ATP indicated that ADP acted as such and not merely as ATP after its dismutation by myokinase. The maximal amount of ATP which could have been formed from ADP could not exceed one third of the original ADP concentration, owing to the equilibrium between ATP, AMP, and ADP, respectively (10). Adenosine monophosphate or AMP plus inorganic pyrophosphate, or inorganic phosphate or pyrophosphate alone were ineffective.
ICTIN
The Adenine
DEPOLYMERIZATION
101
Nucleotide Content of Actin
Since the reversible depolymerization of F-actin by KI occurs within such a short time, it seemed interesting to follow the change of the content of adenine nucleotides and their distribution in the actin during polymerization and depolymerization. Straub and Feuer (8) as well as Laki, Bowen and Clark (11) have shown that] ATP is needed for the polymerization of G-actin. Straub and Feuer found that during this polymerization ATP is dephosphorylated to ADP and free phosphate is liberated; this latter observation was confirmed by Laki and Clark (12). Straub and Feuer assumed that G-actin is ATP-actin and that F-actin is ADP-actin and that during depolymerization of F-actin ATP is resynthesized from ADP, the polymerization being endergonic, while the depolymerization is an exergonic process, the energy of the latter being used for the ATP resynthesis. The ATP, ADP, and AMP content of G-actin, F-actin, and F-actin depolymerized by KI in the presence and absence of ADP was measured (Table I). The results corroborate the finding that ADP is formed from ATP during the polymerization of actin, contrary to the results of Dubuisson and Matthieu (13). G-actin contained mostly ATP, Factin mostly ADP. The total adenine nucleotide content of G-actin was always somewhat lower than that of F-actin. During depolymerization no change occurred in the ratio of the different adenine nucleotides or in the total adenine concentration. If depolymerization was conducted reversibly, enough ADP being added, no ATP formation was observed during this process. The ADP concentration remained constant within experimental errors and no ATP or AMP formation was observed. In these experiments the myokinase was inhibited by the high KI concentration. This is incompatible with the theory of Straub and Feuer. Preparations of three different lots of actin gave identical results. It is worth noting that G- or F-actin does not dephosphorylate ATP, thus containing no ATP-ase, as was also found by the earlier authors. The dephosphorylation observed during polymerization is thus not the result of an unspecific phosphatase action, but is the over-all result of a more complex process. Actin contains myokinase in a concentration high enough to convert a considerable part of the ADP added into AMP and ATP. The activity of this myokinase in 0.6 M KI, however, was practically zero. The myokinase impurity of actin has no effect on the ADP originally pres-
102
ANDREW
Adenmine
Phosphate
G.
SZENT-GY6RGYI
TABLE I for Different
Contents
Treatments
of Actin =
AMP
ADP
ATP
umole/?nl.
rmole/ml.
-Actin No. I, 5 mg./ml. G-a&n F-a&in F-actin + 0.55 M KI for 10 min. F-actin + ADP f 0.55 M KI for 10 min. F-actin + 0.55 M KI 10 min. + boiled for 5 min. + ADP ADP $0.55 M KI Actin No. II, 5.2 mg./ml. G-shin F-actin F-actin + 0.55 M KI for 10 min. F-actin + ADP + 0.55 M KI for 10 min. F-actin + 0.55M KI 10 min. + boiled for 5 min. -I- ADP ADP + 0.55 M KI
Actin No. III, 3.8 mg./ml. G-actin F-actin F-a&in + 0.55 M KI for 10 min. F-a&in + ADP + 0.55M KI for 10 F-2; + 0.55 M KI 10 min. + boiled for 5 min. + ADP ADP + 0.55 M KI
TOtsI adenine nucleotide pnole/ml.
0.000
0.009
0.000
0.005
0.061 0.067
0.045 0.015 0.011.
0.055 0.076 0.083
0.028
0.202
0.015
0.245
0.020 0.011 --
0.195 0.139
0.020 0.003
0.235 0.153
0.015 0.011 0.018
0.015 0.064 0.058
0.040 0.014 0.016
0.070 0.089 0.092
0.025
0.159 I
0.019
0.203
0.028 0.014
0.165 0.108
0.019 0.002
0.212 0.124
0.014 0.011 0.015
0.003 0.039 0.038
0.024 0.004 0.006
0.041 0.054 0.059
0.021
0.157
0.003
0.181
0.037 0.012
0.163 0.117
0.004 0.000
0.204 0.129
.
-- --
--
ent in the actin preparations, showing that this ADP is somehow protected. Storage overnight at 3-5%. decreasesthe adenine nucleotides by about 20% only and does not alter the ratio of ATP, ADP, and AMP. It would have been interesting to see whether ADP, while rendering depolymerization reversible, is dephosphorylated during polymerization, similarly to the ATP originally present. Unfortunately the myokinase impurity of actin made the study of this point impossible. The
ACTIN DEPOLYMERIZATION
103
author was unable to prepare myokinase-free actin. While actin can be liberated from creatinphosphopherase (14) and desaminase by isoelectric precipitation, the myokinase could be eliminated only by prolonged washing of the muscle residue obtained after myosin extraction, during which extraction actin lost its polymerizability. SUMMARY 1. G-a&in obtained from acetone-dried muscle polymerizes in 0.1 M KI after incubation in 0.6 M KI. 2. G-actin prepared by depolymerizing F-a&in by 0.6 M KI does not polymerize in 0.1 M KI. 3. G-actin obtained by depolymerizing F-a&in by 0.6 M KI in the presence of small concentrations of added adenosine triphosphate (ATP), adenosine diphosphate (ADP) or inosine triphosphate (ITP) polymerizes readily in 0.1 M KI. Adenosine monophosphate (AMP), inorganic pyrophosphate, or inorganic phosphate have no such protective action. . 4. Adenosine triphosphate, ADP, and ITP are effective maximally in amounts up to 5 X lo+ mole. 5. The findings of Straub and Feuer that during polymerization ATP is dephosphorylated to ADP is confirmed. 6. Contrary to the findings of Straub and Feuer no synthesis of ATP was observed during depolymerization, not even in the presence of ADP when depolymerization was reversible. REFERENCES 1. STRAUB,F. B., Studies Inst. Med. Chem. Univ. Szeged 2, 3 (1942). 2. ROZSA, G., SZENT-GY~~R~YI,A., AND WYCKOFF, R. W. G., Biochim. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
et Biophys.
Acta 3, 561 (1949). GUBA, F., Nature 166, 439 (1950). SZENT-GY~~RQYI, A. G., AND JOSEPH,R., Arch. Biochem. 31, 90 (1951). KALCHAR,H. M., J. Biol. Chem. 167, 445 (1947). COLOWICK,S. P., AND KALCKAR,H. M., J. Bill. Ch.em. 146, 117 (1943). KALCKAR,H. M., J. Biol. Chem. 167, 461 (1947). STRAUB,F. B., AND FEUER, G., B&him. et Biophys. Acta 4, 455 (1950). LAKI, K., AND CLARIC,A., Personal communication. KALCKAR,H. M., J. Biol. Chem. 146, 127 (1943). LAKI, K., BOWEN, J. W., AND CLARK, A., J. Gen. Physiol. 33, 437 (1950). LAKI, K., AND CLARK, A., Personal communication. DUBUISSON,M., AND MATTHIEU, L., Experientiu 6, 433 (1949). LJWIMOVA, M. N., AND POPOVA, G. M., Doklady Akad. Nauk, S. S. S. R. 66,433 (1949).
the Institute
G. Szent-GySrgyi
GOT Muscle Research at the Marine Woods Hole, Massachusetts Received
November
of Actin 1 Biological
Laboratory,
14, 1950
Actin prepared according to the method of Straub (1) is characterized by its ability to exist in two forms. In salt-free condition it is present in the globular form (G-actin); in the presence of salts it polymerizes into long fibers (F-actin). As shown by Rozsa, Szent-Gyorgyi and Wyckoff (2), this polymerization occurs in two steps: first a number of molecules form larger globules, then these globules unite to form fibers. Straub (1) showed that 0.6 M potassium iodide depolymerizes F-actin. Guba (3) found this effect to be irreversible and instantaneous. In the present paper it is shown that the KI-depolymerization can take place reversibly: data are presented on the changes in the adenine nucleotide content of actin during polymerization and depolymerization. METHODS Actin, adenosine triphosphate (ATP), inosine triphosphate (ITP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were prepared as described previously (4). A stock solution of 6.0 M KI was prepared containing 0.06 M sodium thiosulfate, which prevented the formation of free iodide. The solution was kept in a dark bottle. For the determination of ATP, ADP, and AMP, the enzymatic, differential spectrophotometric method of Kalckar (5) was used, following his description closely. Myokinase (6), desamidase (7), and potato apyrase (7) were prepared as described by Kalckar. The spectrophotometric measurements were made in a Beckman model D. U. spectrophotometer. For the determination of the nucleotides, 4 ml. of the actin solutions was placed in boiling water for 5 min. after each experiment. Then 1 drop of 2 M pH 4.7 sodium acetate buffer was added to the warm actin solutions, which were then filtered, the protein-free liquids being used for the determination. One ml. 1 This
research
was supported
by a grant 97
from
the American
Heart
Association.
98
ANDREW
0. SZENT-QYijRGYI
of the filtrate was diluted with 0.5 ml. water and 1.5 ml. of pH 6.1 0.33 M succinate buffer. The extinction was measured according to Kalckar, his factor being used. The experiments and measurements were made at room temperature, except where otherwise stated. The experiments on G-actin were performed immediately after actin was extracted from the acetone-dried muscle powder. RESULTS
AND DISCUSSION
The E$ect of KI on Actin G-actin, as obtained directly from the acetone-dried muscle powder, polymerizes at almost the same rate and to the same level in 0.1 M KI as in KCl, judging by its viscosity. If this G-a&in is incubated in 0.6 M
83
2
I
I Min.
0
IO
20
30
Fro. 1. Polymerization of G-&in with Polymerization of G-a&in in 0.1 M KCI. 0.1 M KI after incubation in 0.6 M KI for contained 0.0016 M MgClz and 0.0067 M
40
I 50
60
70
and without KI treatment. Empty circles: Semicircles: Polymerization of G-actin in 20 min. at room temperature. Both samples pH 7.0 phosphate buffer.
KI 10 or 20 min. at room temperature and then diluted to contain 0.1 44 KI, the polymerization is still fairly quick and complete, as shown in Fig. 1. If, however, F-actin is subjected to the same treatment, no polymerization takes place. Ten minutes’ incubation of actin in its F-form in 0.6 M KI is more than enough to cause complete and irreversible depolymerization. F-actin incubated with 0.6 M KI for a few minutes depolymerizes completely and will not polymerize again after diluting it to 0.1 M KI concentration. This shows that there is a differ-
ACTIN
99
DEPOLYMERIZATJON
ence between the two G-actins, one obtained directly from the acetonedried muscle powder, the other from F-actin via depolymerization, the latter being extremely labile. The depolymerization of F-actin by KI can be reversed if ATP is added before depolymerization takes place. In this case the G-actin obtained will polymerize again and show the original viscosity of F-actin in about 30 min. The presence of ATP is needed during depolymeriza-
Min.
0
IO
20
30
Fro. 2. Repolymerization of actin. F-a&in was treated with 0.6 M KI for 10 min. and diluted to 0.1 M KI (O-time) in the presence of 0.0016 M MgCl* and 0.0667 A4 pH 7.0 phosphate bufIer. Empty circles: lo+ mole ATP added immediately before addition of 0.6 M KI (ATP concentration calculated for the volume before dilution). Semicircles: 10-s mole ATP added immediately before dilution. Circles with dots: no ATP added. vaDof original F-actin: 0.38.
tion if this process is to be reversible. Adenosine triphosphate added after depolymerization is ineffective (Fig. 2.). The described effect of ATP on actin depolymerization is in accord with the findings of Straub and Feuer (8), according to which depolymerization of F-actin by dialysis against distilled water is reversible only in the presence of ATP. It is also in agreement with the experiments described in the foregoing paper (4), in which depolymerization by urea was found reversible only in the presence of ATP. In both cases ATP was effective only if added before depolymerization. While the
100
ANDREW
G.
SZENT-GYijRGYI
reversible depolymerization by dialysis takes days, and the reversible depolymerization by urea takes hours, the reversible depolymerization by KI can be effected within minutes. This action of ATP was not specific as in the case of urea and contrary to the dialysis experiments of Straub and Feuer (8). Both ADP and ITP were effective in the same amounts as ATP, the lower limit of maximal effect with all three nucleotides lying around 5 X 10m6mole
Fra. 3. Protective action of ATP (vertical semicircles), ADP (black and white semicircies, white top), ITP (black and white semicircles, black top) and AMP (empty circles). Actin was treated for 10 min. with 0.6 M KI in the presence of the nucleotides of diffeferent concentrations (abscissa), added immediately before treatment with KI. Then the solution was diluted to contain 0.1 M KI in the presence of 0.0016 M MgClz and 0.0067 M pH 7.0 phosphate buffer and the viscosity read an hour later (ordinate). qsp of original F-a&in: 0.38.
(Fig. 3). Actin, prepared by Straub’s method, contains myokinase and desamidase, as shown by Laki and Clark (9), making difficult the interpretation of the experiments with ADP. But the fact that ADP was active in as low concentrations as ATP indicated that ADP acted as such and not merely as ATP after its dismutation by myokinase. The maximal amount of ATP which could have been formed from ADP could not exceed one third of the original ADP concentration, owing to the equilibrium between ATP, AMP, and ADP, respectively (10). Adenosine monophosphate or AMP plus inorganic pyrophosphate, or inorganic phosphate or pyrophosphate alone were ineffective.
ICTIN
The Adenine
DEPOLYMERIZATION
101
Nucleotide Content of Actin
Since the reversible depolymerization of F-actin by KI occurs within such a short time, it seemed interesting to follow the change of the content of adenine nucleotides and their distribution in the actin during polymerization and depolymerization. Straub and Feuer (8) as well as Laki, Bowen and Clark (11) have shown that] ATP is needed for the polymerization of G-actin. Straub and Feuer found that during this polymerization ATP is dephosphorylated to ADP and free phosphate is liberated; this latter observation was confirmed by Laki and Clark (12). Straub and Feuer assumed that G-actin is ATP-actin and that F-actin is ADP-actin and that during depolymerization of F-actin ATP is resynthesized from ADP, the polymerization being endergonic, while the depolymerization is an exergonic process, the energy of the latter being used for the ATP resynthesis. The ATP, ADP, and AMP content of G-actin, F-actin, and F-actin depolymerized by KI in the presence and absence of ADP was measured (Table I). The results corroborate the finding that ADP is formed from ATP during the polymerization of actin, contrary to the results of Dubuisson and Matthieu (13). G-actin contained mostly ATP, Factin mostly ADP. The total adenine nucleotide content of G-actin was always somewhat lower than that of F-actin. During depolymerization no change occurred in the ratio of the different adenine nucleotides or in the total adenine concentration. If depolymerization was conducted reversibly, enough ADP being added, no ATP formation was observed during this process. The ADP concentration remained constant within experimental errors and no ATP or AMP formation was observed. In these experiments the myokinase was inhibited by the high KI concentration. This is incompatible with the theory of Straub and Feuer. Preparations of three different lots of actin gave identical results. It is worth noting that G- or F-actin does not dephosphorylate ATP, thus containing no ATP-ase, as was also found by the earlier authors. The dephosphorylation observed during polymerization is thus not the result of an unspecific phosphatase action, but is the over-all result of a more complex process. Actin contains myokinase in a concentration high enough to convert a considerable part of the ADP added into AMP and ATP. The activity of this myokinase in 0.6 M KI, however, was practically zero. The myokinase impurity of actin has no effect on the ADP originally pres-
102
ANDREW
Adenmine
Phosphate
G.
SZENT-GY6RGYI
TABLE I for Different
Contents
Treatments
of Actin =
AMP
ADP
ATP
umole/?nl.
rmole/ml.
-Actin No. I, 5 mg./ml. G-a&n F-a&in F-actin + 0.55 M KI for 10 min. F-actin + ADP f 0.55 M KI for 10 min. F-actin + 0.55 M KI 10 min. + boiled for 5 min. + ADP ADP $0.55 M KI Actin No. II, 5.2 mg./ml. G-shin F-actin F-actin + 0.55 M KI for 10 min. F-actin + ADP + 0.55 M KI for 10 min. F-actin + 0.55M KI 10 min. + boiled for 5 min. -I- ADP ADP + 0.55 M KI
Actin No. III, 3.8 mg./ml. G-actin F-actin F-a&in + 0.55 M KI for 10 min. F-a&in + ADP + 0.55M KI for 10 F-2; + 0.55 M KI 10 min. + boiled for 5 min. + ADP ADP + 0.55 M KI
TOtsI adenine nucleotide pnole/ml.
0.000
0.009
0.000
0.005
0.061 0.067
0.045 0.015 0.011.
0.055 0.076 0.083
0.028
0.202
0.015
0.245
0.020 0.011 --
0.195 0.139
0.020 0.003
0.235 0.153
0.015 0.011 0.018
0.015 0.064 0.058
0.040 0.014 0.016
0.070 0.089 0.092
0.025
0.159 I
0.019
0.203
0.028 0.014
0.165 0.108
0.019 0.002
0.212 0.124
0.014 0.011 0.015
0.003 0.039 0.038
0.024 0.004 0.006
0.041 0.054 0.059
0.021
0.157
0.003
0.181
0.037 0.012
0.163 0.117
0.004 0.000
0.204 0.129
.
-- --
--
ent in the actin preparations, showing that this ADP is somehow protected. Storage overnight at 3-5%. decreasesthe adenine nucleotides by about 20% only and does not alter the ratio of ATP, ADP, and AMP. It would have been interesting to see whether ADP, while rendering depolymerization reversible, is dephosphorylated during polymerization, similarly to the ATP originally present. Unfortunately the myokinase impurity of actin made the study of this point impossible. The
ACTIN DEPOLYMERIZATION
103
author was unable to prepare myokinase-free actin. While actin can be liberated from creatinphosphopherase (14) and desaminase by isoelectric precipitation, the myokinase could be eliminated only by prolonged washing of the muscle residue obtained after myosin extraction, during which extraction actin lost its polymerizability. SUMMARY 1. G-a&in obtained from acetone-dried muscle polymerizes in 0.1 M KI after incubation in 0.6 M KI. 2. G-actin prepared by depolymerizing F-a&in by 0.6 M KI does not polymerize in 0.1 M KI. 3. G-actin obtained by depolymerizing F-a&in by 0.6 M KI in the presence of small concentrations of added adenosine triphosphate (ATP), adenosine diphosphate (ADP) or inosine triphosphate (ITP) polymerizes readily in 0.1 M KI. Adenosine monophosphate (AMP), inorganic pyrophosphate, or inorganic phosphate have no such protective action. . 4. Adenosine triphosphate, ADP, and ITP are effective maximally in amounts up to 5 X lo+ mole. 5. The findings of Straub and Feuer that during polymerization ATP is dephosphorylated to ADP is confirmed. 6. Contrary to the findings of Straub and Feuer no synthesis of ATP was observed during depolymerization, not even in the presence of ADP when depolymerization was reversible. REFERENCES 1. STRAUB,F. B., Studies Inst. Med. Chem. Univ. Szeged 2, 3 (1942). 2. ROZSA, G., SZENT-GY~~R~YI,A., AND WYCKOFF, R. W. G., Biochim. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
et Biophys.
Acta 3, 561 (1949). GUBA, F., Nature 166, 439 (1950). SZENT-GY~~RQYI, A. G., AND JOSEPH,R., Arch. Biochem. 31, 90 (1951). KALCHAR,H. M., J. Biol. Chem. 167, 445 (1947). COLOWICK,S. P., AND KALCKAR,H. M., J. Bill. Ch.em. 146, 117 (1943). KALCKAR,H. M., J. Biol. Chem. 167, 461 (1947). STRAUB,F. B., AND FEUER, G., B&him. et Biophys. Acta 4, 455 (1950). LAKI, K., AND CLARIC,A., Personal communication. KALCKAR,H. M., J. Biol. Chem. 146, 127 (1943). LAKI, K., BOWEN, J. W., AND CLARK, A., J. Gen. Physiol. 33, 437 (1950). LAKI, K., AND CLARK, A., Personal communication. DUBUISSON,M., AND MATTHIEU, L., Experientiu 6, 433 (1949). LJWIMOVA, M. N., AND POPOVA, G. M., Doklady Akad. Nauk, S. S. S. R. 66,433 (1949).