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Adenosine triphosphate from insect muscle
Adenosine Triphosphate from Insect Muscle J. H. Calaby From the Division of Entomology, Commonwealth Scientijic and Indwtrial Research Organization, Canberra, Australia Received December 8, 1950
In 1948 Albaum and Kletzkin isolated a nucleotide from the insect, Drosophila melunogaster, and concluded from the results of a series of chemical and enzymatic tests that its composition was identical with that of mammalian adenosine triphosphate (ATP). In view of the fact that the material investigated by Albaum and Kletzkin was relatively impure, and the availability to me of insect ATP in a very pure state isolated in the course of an investigation on the enzyme systems of insect muscle, it seemed worthwhile to repeat the earlier work on the composition of insect ATP. This paper comprises a brief summary of the details of a comparison of pure rabbit and insect ATP’s which completely confirmed the conclusion of Albaum and Kletzkin regarding the identity of the two compounds. MATERIALS AND METHODS The insect used was the locust, Gastrimargus musks Fabr., which was collected in large numbers in the field at Canberra. The hind femora and thoraces (from which the gut had been withdrawn) were removed under ether anesthesia, and ground with trichloroacetic acid, either with sand in a mortar, or in a Waring Blendor. The dibarium salt of the ATP was isolated and purified by repeated precipitation by Kerr’s (1941) method without modification. Rabbit ATP was prepared in the same manner. Air-dried powders were used for the assay of N, total, labile, and inorganic P, and water of crystallization. All other tests were done on a number of stock solutions of the Na salt, of various concentrations, prepared by passing an acid solution of the Ba salt through a column of Amberlite IR-100, by the method recommended by Polis and Meyerhof (1947). The concentrations of these solutions were determined accurately by measuring the’ labile P.
294
ATP FROM INSECT MUSCLE
295
Details of the Methods Used Water of Crystallization. Determined as the loss in weight of a 19-mg. sample after heating for 4 hr. at 190°C. over P,OS in vaczw. Total P. Five-mg. samples were digested in H&O4 and, after dilution, the BaSO, was filtered off and P determined in aliquots of the final solution by the method of Fiske and SubbaRow (1925) using a photoelectric calorimeter with a red filter. Inorganic P. Determined on 5-mg. samples dissolved in a small amount of icecold 1 N HCl. After solution the Ba was precipitated with Na&SO+ the BaSObw&9 filtered off and washed, and P was determined in the same manner as total P. Labile P. Five-mg. samples were hydrolyzed in 1 N HCl for 11-12 min. After the precipitation of the Ba with Na$O+ the procedure followed was the same as for total P determinations. A hydrolysis time of 11-12 min., rather than the conventional 7 min., was found to be necessary for the complete breakdown of the labile P [cf. also, Bailey (1949), who recommended hydrolysis for 9-10 min.]. N. Determined by Nesslerization in aliquots of the solutions prepared for total P estimation, using the reagent of Vanselow (1940). Measurements were made in a Hilger “Spekker” Absorptiometer using an Ilford spectrum violet filter 691. Purine. Estimated in the Beckman spectrophotometer at 265 rnp using adenine compounds as standards. The purine was identified by its ultraviolet absorption spectrum measured at pH 7. Pentose. Mejbaum’s (1939) method, as modified by Le Page and Umbreit (1947), was used. The samples were read in the Beckman spectrophotometer at 660 mp. The pentose was identified by color development time curves in the orcinol-pentose reaction, according to the method of Albaum and Umbreit (1947). Pyrophosphate Linkages. Adenosine triphosphate solutions were treated with pyrophosphatases under optimal conditions. The enzymes used were myosin brepared by Bailey’s (1942) method], myosin plus myokinase [prepared by the method of Colowick and Kalckar (1943)], and insect Mg-activated apyrase [Gilmour (1948)]. Position of Phosphafes cm Pentose Component. Shown by hydrolysis rate as measured by the color development curves in the orcinol-pentose reaction. Further proof was given by a modification of a test used by Albaum and Kletrkin (1948), based on the specificity of muscle deaminase to adenosine-5-phosphoric acid. Adenosine triphosphate solutions were dephosporylated by the insect apyrase, then incubated with myosin (acting as adenylic acid deaminase). Parallel tests were done with pure commercial samples of muscle adenosine-5-phosphoric acid and yeast adenosine-3-phosphoric acid. Samples were withdrawn at intervals, the reaction was stopped with CChCOOH, and, after filtering, the density was measured on a suitably diluted aliquot at 265 mp in the Beckman spectrophotometer.
RESULTS
Analytical data obtained for the ATP samples are given in Table I. The ultraviolet absorption spectrum of insect ATP shows that the purine is adenine. The curves obtained for rabbit and insect ATP’s agree in contour with each other and with those given by Dounce et al.
296
J. H. CALABY
TABLE Analysis
I
of Adenosine Triphosphute Found
-
-Adenine Ribose Inorganic Total P PI Labile P PI Nitrogen Water of
P (corrected for inorganic
Rabbit
.-
Theoretical for LaKAoHnOt1NrPa.6Hz0 Ins&
15%
155
23
16.8 0.35
17.2 0.35
16.9 0.0
10.46
10.36
10.49
7.07 7.85 11.3
7.10 7.99 11.3
6.99 7.91 12.2
(corrected for inor@nic
crystallization
-
-
(1948) and Kalckar (1947B). The absorption maximum is at 260 rnp. Figure 1 shows the absorption spectrum of insect ATP. Values obtained for the absorption coefficient [WON,,= (l/C> X (log,, IO/I)] were 1.62 X lo4 for rabbit ATP and 1.59 X lo4 for insect x lO-5 Mol.
220
230
240
250
260
Wave length,
FIQ. 1. Ultraviolet
270
280
290
mp
absorption spectrum of insect ATP. Done tration of 3.59 X lo+ M.
at
pH 7 at a concen-
ATP
FROM
INSECT
MUSCLE
297
ATP, in good agreement with Kalckar (19478) and Dounce et al. who obtained 1.6 X lo4 and 1.62 X 104, respectively, for rabbit ATP. Time curves in the orcinol-pentose reaction with rabbit and insect ATP’s were identical in equimolar concentration. The fact that insect ATP is readily hydrolyzed by pyrophosphatases shows that the labile P is of the pyrophosphate type. Figure 2 shows the effect of myosin alone and myosin plus myokinase on insect ATP.
Time, minutes
FIQ. 2. Hydrolysis of insect ATP by pyrophosphataaes. Lower curve, myosin alone; upper curve, myosin + myokinaae. Each tube contained 4 ml. myosin solution or myosin + myokinase, buffered at pH 9; 2 ml. ATP solution (containing approximately 300 ~cg.labile P/ml.); and 0.2 ml. 0.1 M CaCle. Incubatedat 37°C. Samples of I ml. were withdrawn at intervals.
Determinations of the Michaelis constants of insect Mg-activated apyrase using both rabbit and insect ATP as substrates have shown that insect and rabbit ATP’s are hydrolyzed with about equal facility (Gilmour and Calaby, unpublished data). The action of muscle deaminase on adenosine-5phosphoric acid, adenosine-3-phosphoric acid, and dephosphorylated rabbit ATP and insect ATP is illustrated in Fig. 3. It is apparent that the deaminase
298
J. H. CALABY
0 % A 6
Adenosine-5-phosphoric Dephosphoryloted insect Dephosphoryloted robbit Adenosine-3- phosphoric
I
IO Time, minutes
acid ATP ATP acid
l 30
FIQ. 3. The effect of muscle myosin-deaminase on adenylic acids from various sources. 0 adenosine&phosphoric acid; l adenosine&phosphoric acid; A rabbit ATP after preliminary dephosphorylation by an insect apyrase; X insect ATP after preliminary dephosphorylation by insect apyrase. Deamination was measured by the decrease in optical density at 265 rnp in a Beckman spectrophotometer. The lower rate of deamination in the case of the two dephosphorylated ATP’s was the result of using lower substrate concentrations in these experiments. The percentage deaminations were about the same as in the case of the 5-adenylic acid. Each tube contained 1.5 ml. apyrase, 0.75 ml. ATP (containing approximately 300 pg. labile P/ml.) or 0.75 ml. adenylic acid (containing approximately 5 mg. adenylic acid/ml.), and 0.19 ml. 0.026 N MgClz. After incubation for 2.5 hr. at pH 8 and 42°C the pH was reduced to 6.5 and 1 ml. myosin solution was added. Samples were withdrawn at intervals for analysis in the spectrophotometer.
against the 3-adenylic acid, but produced a rapid deamination of the 5-adenylic acid and the rabbit and insect ATP derivatives. From this it was concluded that the phosphates of the insect ATP are located on the Spositions of the ribose moiety. wa,s ineffective
ATP FROM
INSECT
MUSCLE
299
ACKNOWLEDGMENTS
The author is indebted to Mr. D. Gilmour for much helpful discussion and to Mr. D. L. McIntosh who prepared the original samples with great skill. SUMMARY
Adenosine triphosphates isolated from insect and rabbit muscle have been compared on the basis of a number of chemical, physical, and enzymatic tests, and the insect material is shown to be identical with that obtained from rabbit. REFERENCES 1. ALBAUM, H. G., AND KLETZKIN, M., Arch. Biochem. 16, 333 (1948). 2. ALBAW, H. G., AND UMBREIT, W. W., J. Biol. Chem. 167, 369 (1947). 3. BAILEY, K., Biochem. J. 36, 121 (1942). 4. BAILEY, K., Biochem. J. 45, 479 (1949). 5. COLOWICK, S. P., AND KALCKAR, H. M., J. Biol. Chem. 146, 117 (1943). 6. DOUNCE, A. L., ROTHSTEIN, A., BEYER, G. T., MEIER, R., AND FREER, R., J Biol. Chem. 174, 361 (1948). 7. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 8. GILMOUR, D., J. Biol. Chem. 175, 477 (1948). 9. KALCKAR, H. M., J. Biol. Chem. 167, 445 (19478). 10. KALCKAR, H. M., Symp. Sot. Exptl. Biol. 1, 38 (1947B). 11. KERR, S. E., J. Biol. Chem. 139, 121 (1941). 12. LE PAGE, G. A., AND UMBF~EIT, W. W., in Manometric Techniques and Related Methods for the Study of Tissue Metabolism, edited by UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., p. 159. Burgess Publishing Co.
Minneapolis, 1947. 13. MEJBAUM, W., 2. physiol. Chem. 258, 117 (1939). 14. POLIS, B. D., AND MEYERHOF, O., J. Biol. Chem. 169, 389 (1947). 15. VANSELOW, A. P., Ind. Eng. Chem., Anal. Ed. 12, 516 (1940).
In 1948 Albaum and Kletzkin isolated a nucleotide from the insect, Drosophila melunogaster, and concluded from the results of a series of chemical and enzymatic tests that its composition was identical with that of mammalian adenosine triphosphate (ATP). In view of the fact that the material investigated by Albaum and Kletzkin was relatively impure, and the availability to me of insect ATP in a very pure state isolated in the course of an investigation on the enzyme systems of insect muscle, it seemed worthwhile to repeat the earlier work on the composition of insect ATP. This paper comprises a brief summary of the details of a comparison of pure rabbit and insect ATP’s which completely confirmed the conclusion of Albaum and Kletzkin regarding the identity of the two compounds. MATERIALS AND METHODS The insect used was the locust, Gastrimargus musks Fabr., which was collected in large numbers in the field at Canberra. The hind femora and thoraces (from which the gut had been withdrawn) were removed under ether anesthesia, and ground with trichloroacetic acid, either with sand in a mortar, or in a Waring Blendor. The dibarium salt of the ATP was isolated and purified by repeated precipitation by Kerr’s (1941) method without modification. Rabbit ATP was prepared in the same manner. Air-dried powders were used for the assay of N, total, labile, and inorganic P, and water of crystallization. All other tests were done on a number of stock solutions of the Na salt, of various concentrations, prepared by passing an acid solution of the Ba salt through a column of Amberlite IR-100, by the method recommended by Polis and Meyerhof (1947). The concentrations of these solutions were determined accurately by measuring the’ labile P.
294
ATP FROM INSECT MUSCLE
295
Details of the Methods Used Water of Crystallization. Determined as the loss in weight of a 19-mg. sample after heating for 4 hr. at 190°C. over P,OS in vaczw. Total P. Five-mg. samples were digested in H&O4 and, after dilution, the BaSO, was filtered off and P determined in aliquots of the final solution by the method of Fiske and SubbaRow (1925) using a photoelectric calorimeter with a red filter. Inorganic P. Determined on 5-mg. samples dissolved in a small amount of icecold 1 N HCl. After solution the Ba was precipitated with Na&SO+ the BaSObw&9 filtered off and washed, and P was determined in the same manner as total P. Labile P. Five-mg. samples were hydrolyzed in 1 N HCl for 11-12 min. After the precipitation of the Ba with Na$O+ the procedure followed was the same as for total P determinations. A hydrolysis time of 11-12 min., rather than the conventional 7 min., was found to be necessary for the complete breakdown of the labile P [cf. also, Bailey (1949), who recommended hydrolysis for 9-10 min.]. N. Determined by Nesslerization in aliquots of the solutions prepared for total P estimation, using the reagent of Vanselow (1940). Measurements were made in a Hilger “Spekker” Absorptiometer using an Ilford spectrum violet filter 691. Purine. Estimated in the Beckman spectrophotometer at 265 rnp using adenine compounds as standards. The purine was identified by its ultraviolet absorption spectrum measured at pH 7. Pentose. Mejbaum’s (1939) method, as modified by Le Page and Umbreit (1947), was used. The samples were read in the Beckman spectrophotometer at 660 mp. The pentose was identified by color development time curves in the orcinol-pentose reaction, according to the method of Albaum and Umbreit (1947). Pyrophosphate Linkages. Adenosine triphosphate solutions were treated with pyrophosphatases under optimal conditions. The enzymes used were myosin brepared by Bailey’s (1942) method], myosin plus myokinase [prepared by the method of Colowick and Kalckar (1943)], and insect Mg-activated apyrase [Gilmour (1948)]. Position of Phosphafes cm Pentose Component. Shown by hydrolysis rate as measured by the color development curves in the orcinol-pentose reaction. Further proof was given by a modification of a test used by Albaum and Kletrkin (1948), based on the specificity of muscle deaminase to adenosine-5-phosphoric acid. Adenosine triphosphate solutions were dephosporylated by the insect apyrase, then incubated with myosin (acting as adenylic acid deaminase). Parallel tests were done with pure commercial samples of muscle adenosine-5-phosphoric acid and yeast adenosine-3-phosphoric acid. Samples were withdrawn at intervals, the reaction was stopped with CChCOOH, and, after filtering, the density was measured on a suitably diluted aliquot at 265 mp in the Beckman spectrophotometer.
RESULTS
Analytical data obtained for the ATP samples are given in Table I. The ultraviolet absorption spectrum of insect ATP shows that the purine is adenine. The curves obtained for rabbit and insect ATP’s agree in contour with each other and with those given by Dounce et al.
296
J. H. CALABY
TABLE Analysis
I
of Adenosine Triphosphute Found
-
-Adenine Ribose Inorganic Total P PI Labile P PI Nitrogen Water of
P (corrected for inorganic
Rabbit
.-
Theoretical for LaKAoHnOt1NrPa.6Hz0 Ins&
15%
155
23
16.8 0.35
17.2 0.35
16.9 0.0
10.46
10.36
10.49
7.07 7.85 11.3
7.10 7.99 11.3
6.99 7.91 12.2
(corrected for inor@nic
crystallization
-
-
(1948) and Kalckar (1947B). The absorption maximum is at 260 rnp. Figure 1 shows the absorption spectrum of insect ATP. Values obtained for the absorption coefficient [WON,,= (l/C> X (log,, IO/I)] were 1.62 X lo4 for rabbit ATP and 1.59 X lo4 for insect x lO-5 Mol.
220
230
240
250
260
Wave length,
FIQ. 1. Ultraviolet
270
280
290
mp
absorption spectrum of insect ATP. Done tration of 3.59 X lo+ M.
at
pH 7 at a concen-
ATP
FROM
INSECT
MUSCLE
297
ATP, in good agreement with Kalckar (19478) and Dounce et al. who obtained 1.6 X lo4 and 1.62 X 104, respectively, for rabbit ATP. Time curves in the orcinol-pentose reaction with rabbit and insect ATP’s were identical in equimolar concentration. The fact that insect ATP is readily hydrolyzed by pyrophosphatases shows that the labile P is of the pyrophosphate type. Figure 2 shows the effect of myosin alone and myosin plus myokinase on insect ATP.
Time, minutes
FIQ. 2. Hydrolysis of insect ATP by pyrophosphataaes. Lower curve, myosin alone; upper curve, myosin + myokinaae. Each tube contained 4 ml. myosin solution or myosin + myokinase, buffered at pH 9; 2 ml. ATP solution (containing approximately 300 ~cg.labile P/ml.); and 0.2 ml. 0.1 M CaCle. Incubatedat 37°C. Samples of I ml. were withdrawn at intervals.
Determinations of the Michaelis constants of insect Mg-activated apyrase using both rabbit and insect ATP as substrates have shown that insect and rabbit ATP’s are hydrolyzed with about equal facility (Gilmour and Calaby, unpublished data). The action of muscle deaminase on adenosine-5phosphoric acid, adenosine-3-phosphoric acid, and dephosphorylated rabbit ATP and insect ATP is illustrated in Fig. 3. It is apparent that the deaminase
298
J. H. CALABY
0 % A 6
Adenosine-5-phosphoric Dephosphoryloted insect Dephosphoryloted robbit Adenosine-3- phosphoric
I
IO Time, minutes
acid ATP ATP acid
l 30
FIQ. 3. The effect of muscle myosin-deaminase on adenylic acids from various sources. 0 adenosine&phosphoric acid; l adenosine&phosphoric acid; A rabbit ATP after preliminary dephosphorylation by an insect apyrase; X insect ATP after preliminary dephosphorylation by insect apyrase. Deamination was measured by the decrease in optical density at 265 rnp in a Beckman spectrophotometer. The lower rate of deamination in the case of the two dephosphorylated ATP’s was the result of using lower substrate concentrations in these experiments. The percentage deaminations were about the same as in the case of the 5-adenylic acid. Each tube contained 1.5 ml. apyrase, 0.75 ml. ATP (containing approximately 300 pg. labile P/ml.) or 0.75 ml. adenylic acid (containing approximately 5 mg. adenylic acid/ml.), and 0.19 ml. 0.026 N MgClz. After incubation for 2.5 hr. at pH 8 and 42°C the pH was reduced to 6.5 and 1 ml. myosin solution was added. Samples were withdrawn at intervals for analysis in the spectrophotometer.
against the 3-adenylic acid, but produced a rapid deamination of the 5-adenylic acid and the rabbit and insect ATP derivatives. From this it was concluded that the phosphates of the insect ATP are located on the Spositions of the ribose moiety. wa,s ineffective
ATP FROM
INSECT
MUSCLE
299
ACKNOWLEDGMENTS
The author is indebted to Mr. D. Gilmour for much helpful discussion and to Mr. D. L. McIntosh who prepared the original samples with great skill. SUMMARY
Adenosine triphosphates isolated from insect and rabbit muscle have been compared on the basis of a number of chemical, physical, and enzymatic tests, and the insect material is shown to be identical with that obtained from rabbit. REFERENCES 1. ALBAUM, H. G., AND KLETZKIN, M., Arch. Biochem. 16, 333 (1948). 2. ALBAW, H. G., AND UMBREIT, W. W., J. Biol. Chem. 167, 369 (1947). 3. BAILEY, K., Biochem. J. 36, 121 (1942). 4. BAILEY, K., Biochem. J. 45, 479 (1949). 5. COLOWICK, S. P., AND KALCKAR, H. M., J. Biol. Chem. 146, 117 (1943). 6. DOUNCE, A. L., ROTHSTEIN, A., BEYER, G. T., MEIER, R., AND FREER, R., J Biol. Chem. 174, 361 (1948). 7. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 8. GILMOUR, D., J. Biol. Chem. 175, 477 (1948). 9. KALCKAR, H. M., J. Biol. Chem. 167, 445 (19478). 10. KALCKAR, H. M., Symp. Sot. Exptl. Biol. 1, 38 (1947B). 11. KERR, S. E., J. Biol. Chem. 139, 121 (1941). 12. LE PAGE, G. A., AND UMBF~EIT, W. W., in Manometric Techniques and Related Methods for the Study of Tissue Metabolism, edited by UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., p. 159. Burgess Publishing Co.
Minneapolis, 1947. 13. MEJBAUM, W., 2. physiol. Chem. 258, 117 (1939). 14. POLIS, B. D., AND MEYERHOF, O., J. Biol. Chem. 169, 389 (1947). 15. VANSELOW, A. P., Ind. Eng. Chem., Anal. Ed. 12, 516 (1940).