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Free and bound nucleotides in frog and mammalian muscle
ARCHIVES
OF
Free
BIOCHEMISTRY
and
JACOB
AND
Bound
Nucleotides
SACKS,
Department
122,
BIOPHYSICS
591-593
(1967)
in Frog
and
Mammalian
WILLIAM D. DUCKETT, SALLYE MARGARET SCHAEFER
oj Chemistry, Received
University April
of Arkansas,
13, 1967;
accepted
Fayetteville, June
Muscle’
MORGAN,
Arkansas
ASD
72701
23, 1967
The Atp of muscle can be separated into three fractions by differential extraction of the powered frozen muscle. A fraction firmly bound to protein, equivalent to 5-6 moles/mole of myosin in bullfrogs, and to more than twice this amount in cats, is considered to act as the bridge between myosin and actin necessary to account for the structure of resting striated muscle shown by the electron microscope. A second fraction is also considered to be protein-bound, but fairly readily dissociable therefrom. More ATP is found unbound to protein in bullfrog than in cat muscle. The ADP in both species is essentially all protek-bound and is present in an amount equivalent to t,he 1 mole/mole of F-actin monomer that has been found for the isolated protein. Tetanic contraction of 15 seconds duration does not lead to any significant change in relations between free and bound nucleotide derivatives.
Extraction of frozen, powdered mammalian muscle wit.h 65 % ethanol at -20” has been used to dissolve the free nucleotide derivatives and thus separate them from protein-bound material (l), rendered soluble by treatment of the extraction residue with trichloroacetic acid. Practically all the ADP and much of the ATP are found in the protein-bound fraction. The amount of bound ADP corresponds to the known actin content of mammalian muscle (2) and the well-established capacity of the fibrous form of this protein to bind ADP (2, 3). The only protein present in sufficient quantity to account for the bound ATP is myosin, and this only on the basis that one molecule of myosin binds several molecules of ATP. These data served as the basis for postulating that the bound ATP acts as the bridge between myosin and actin needed to account for the structure of resting muscle shown by the electron microscope. It] was also postu1 This work was supported AM 07273 from the National ritis and Metabolic diseases, IYational Science Folmdation partment, for an Undergradrlatc pation Program.
lated that this binding of ATP to myosin conforms to the requirements of an allosteric inhibitor of the ATPase action of myosin, by preventing the change in conformation about the active center necessary for the enzymic activity to be manifested (1). This type of inhibition can account for the failure to find direct evidence for the breakdown of ATP in cont’racting muscle (4, 5), or indirect evidence in the form of lack of the equilibration of 32P between phosphocreatine and terminal P of ATP that would be expected to result from the resynthesis of ATP by the creatine kinase reaction (6, 8). The present experiments were undertaken with two objectives. One was to compare amphibian with mammalian muscle with respect to the quant’itative relations between free and bound ADP and ATP. The other was to explore the possibility that the ATP was present in three functional compartments, rather than two, of which one might undergo dephosphorylation in eontraction and resynthesis by the creatine kinase reaction. If such were the case, there was the possibility that a tracer experiment would show equilibration of the isotope between phosphocreatine and the terminal P of the
in part by grant Institllte of Arthand in part by a grant to this deResearch Partici591
592
;
SACKS
ATP in this compartment only. Data were obtained that can be interpreted in terms of three such functional compartments. The results of the tracer experiments, to be reported later, do not indicate any tendency toward the establishment of isotopic equilibrium between the phosphocreatine and the terminal P of the ATP present in any one of the three compartments. The data on frog muscle show the same qualitative distribution of the ATP and ADP as in mammalian muscle. Quantitatively, very much less ATP is protein-bound in the frog muscle. EXPERIMENTAL
ET
musculature of the hind legs was used, with particular precautions to remove bone, cartilage, and tendons. The frozen muscles were powdered in a tissue crusher chilled with dry ice and treated in the Waring Blendor with 10 volumes of 80% ethanol saturated with EDTA, chilled to -2O”, let stand with occasional shaking for 10 minutes, and centrifuged at -20”. The residue was extracted twice with 5 volumes each of the 80% ethanol, then three times with 65y0 ethanol containing 0.001 M EDTA, and finally with 10% trichloroacetic acid. The separate extracts were treated as in the previous work (1) to remove small amounts of proteinaceous material in the ethanolic extracts, and with ether t)o remove trichloroacetic acid. The P compounds were absorbed on columns of Dowex-I resin. Elution of the extracts from the bullfrog muscles was begun with 0.002 N HCl, which would have eluted any AMP present. None was found, as had been the situation previously with mammalian muscle. ADP was eluted with 0.01 N HCl, and ATP with 0.01 N HCl + 0.04 M KCl, as before. Measurements of ADP and ATP in the various fractions of the eluates were made in terms of absorbance at 260 rnp.
PROCEDURE
The experiments on cats were carried out as in the earlier work (1) by pouring the freezing mixture over one gastocnemius muscle 15 seconds after the commencement of tetanic contraction under load, then freezing the resting muscle. Bullfrogs (Rana catesbiana) rather than grass frogs (Rana pipiens) were used in order to have muscle samples from one animal comparable in quantity to those obtained from the cats. The bullfrogs were stored in the cold room, allowed to warm up slowly overnight to room temperature-these experiments were done during the summer monthsand decerebrated with the point of a scapel. After recovery from the shock of the operation, one sciatic plexus was exposed high in the body cavity, stimulating electrodes were placed, and the nerve was cut proximal to the electrodes. The animal was then placed in a trough longer than the extended length, the tetanizing current was started, and 15 seconds later a large quantity of the freezing mixture, enough to freeze the entire animal, was portred into the trough. The entire
RESULTS
Muscle
Cat,
resting
Cat,
tetanized
Frog,
resting
Frog,
tetanized
OF ATP
AND
I AND
ADP
IN
MUSCLES
ATP FL%+
0.37 (0.03-O. 58) 0.39 (0.15-1.40) 0.87 (0.48-l. 76) 0.71 (0.32-l. 75)
a Values are rmoles/g, mean and range. * Free: extracted by 80% ethanol. Dissociable: ethanol; in trichloroacetic acid extract.
DISCUSSION
The data on the distribution of ADP and ATP in the three extraction media are summarized in Table I. In one of the experiments on cats, and in two of those on bullfrogs, faint traces of ADP were found in the 80% ethanolic extracts. The content of both ADP and ATP is significantly lower in bullfrog muscle than in cat muscle. The actin content of bullfrog muscle is not known, but Dolp (9) has shown
TABLE DISTRIBUTION
AL.
Dissociable
1.33 (0.88-l .64) 1.16 (0.26-2.12) 2.04 (1.18-3.02) 1.89 (0.74-3.40)
extracted
Bound
2.93 (1.61-3.99) 2.57 (1.66-3.93) 0.84 (0.20-l. 52) 0.90 (0.21-1.40)
by 65y0 ethanol.
ADP,
bound
No. expts.
0.53 (0.30-0.57) 0.44 (0.28-0.56) 0.33 (0.25-0.40) 0.37 (0.26-0.42)
Bound:
not
13 13 8 8
extracted
by
FREE
AND
BOUND
ADP
that the molecular weight of the monomer is the same as that reported by others for material from mammalian sources. On this basis, the present data suggest that all the actin and all the ADP in bullfrog muscle, as in mammalian, are present as the complex of F-actin with ADP, and that the ADP content can be taken as a measure of t’he actin content. While the data in viva and in vitro with respect to the protein binding of ADP are in good agreement, the same does not hold for ATP. The determination with isolated myosin is complicated by the ATPase action which appears under these conditions. Two reports (10, 11) give values which extrapolate to a value of 1 mole/mole of myosin, a value far lower than indicated by the findings in viva. The average amount of bound ATP in the bullfrog muscles corresponds to 5 or 6 moles/mole of myosin, on the assumptions that the myosin content of the muscles of this species is not significantly different from the 10% reported for rabbit muscle (12) and that the molecular weight is of the same order as that generally given for mammalian muscle, about 600,000. The value in the cat muscle corresponds to at least twice this molar ratio. The finding that much of the ATP not firmIy bound to protein is not extractable b-J 80% ethanol in the presence of EDTA, a medium in which the Na and K salts of ATP are freely soluble, suggests that the fraction extracted by 65 % ethanol may also be protein-bound, in a form more readily dissociable than that which requires denaturation of the protein to render the ATP extractable. The amount of this fraction is so great, especially in the bullfrog muscle, that t,he possibility needs to be considered that this fraction also is bound to myosin, in some other way than that which is firmly bound. This fraction is listed in the table as dissociable. Tetanic contraction does not alter significantly the distribution of ATP between free, dissociable, and firmly bound fractions. This situation is compatible with either the sliding filament theory or the one previously proposed (13), that the contraction is initiated by the breaking of the bridges on the
AND
ATP
IN
MUSCLE
593
actin side. The sliding filament theory does not require that the mat’erial constituting t’he bridge separate from both proteins in the breaking of the bridge. In the version proposed by Davies (14), retention of one ATP per H-meromyosin to the protein is specifically called for during the breaking and making of the bridge. The amount of firmly bound ATP is sufficient to allow one ATP-myosin bridge in the frog muscle, and two or more in the cat muscle, to each of the six actin filaments that the electron micrographs show to surround each myosin filament. In the theory proposed (13) as an alternate to the sliding filament, the rupture of the bond between the ATP and actin that allows the actin to pass into the disordered state does not require any breaking of the bonds between ATP and myosin. The present data do not permit a choice between the two theories; it does need to be pointed out that neither has an obligatory requirement for conversion of the bound ATP to ADP. REFERENCES 1. SACKS, J., MURPHREE, S., AND BROWN, It., Arch. Biochem. f?iophys.113,97 (1966). 2. HASSELRACH, W., B&him. Biophys. Ada 26, 652 (1957). 3. MARTONOSI, A., GOUVEA, M. A., AND GERGELY J., ./. Hiok Chem. 236, 1707 (1960); BARANY, M., NAGY, B., FINKELMAN, F., AND CHRAMBACH, A., J. Biol. Chem. 236, 2917 (1961). 4. MOMMAERTS, W. F. H. M., -am. J. Physiol. lE2, 585 (1955). 5. CIRLSON, F. D., AND SIGER, A.,J. Gen. Physiol. 44, 33 (1960). 6. FLECKENSTEIN, A., JANKE, J., LECHNER, M., I\ND BAuER, (i., i’jtuger’s drch. 269, 246 (1954). 7. DIXON, G. J., BND SACKS, J., ilm. J. Physiol. 193, 129 (1958). 8. Sacks, J., .IND CLELAND, M., Am. J. Physiol. 198, 300 (1960). 9. DOLP, R. M., Arch. Hiochem. Biophys. 113, 20 (1966). 10. NANNINGA, L. B., AND MOMM.IERTS, w. F. H. M., Proc. AVatl. Acad. Sci. U.S. 46, 1155 (1960). 11. MARTONOSI, A., AND MEYER, H., J. Biol. Chem. 239, 640 (1954). 12. HASSELBACH, w., AND SCHNEIDER, G., l?iothem. Z. 321, 462 (1951). 13. SACKS, J., Perspectives Biol. Med. 7,285 (1964). 14. D.~vIs, It. E., Nab-e 109, 1068 (1963).
OF
Free
BIOCHEMISTRY
and
JACOB
AND
Bound
Nucleotides
SACKS,
Department
122,
BIOPHYSICS
591-593
(1967)
in Frog
and
Mammalian
WILLIAM D. DUCKETT, SALLYE MARGARET SCHAEFER
oj Chemistry, Received
University April
of Arkansas,
13, 1967;
accepted
Fayetteville, June
Muscle’
MORGAN,
Arkansas
ASD
72701
23, 1967
The Atp of muscle can be separated into three fractions by differential extraction of the powered frozen muscle. A fraction firmly bound to protein, equivalent to 5-6 moles/mole of myosin in bullfrogs, and to more than twice this amount in cats, is considered to act as the bridge between myosin and actin necessary to account for the structure of resting striated muscle shown by the electron microscope. A second fraction is also considered to be protein-bound, but fairly readily dissociable therefrom. More ATP is found unbound to protein in bullfrog than in cat muscle. The ADP in both species is essentially all protek-bound and is present in an amount equivalent to t,he 1 mole/mole of F-actin monomer that has been found for the isolated protein. Tetanic contraction of 15 seconds duration does not lead to any significant change in relations between free and bound nucleotide derivatives.
Extraction of frozen, powdered mammalian muscle wit.h 65 % ethanol at -20” has been used to dissolve the free nucleotide derivatives and thus separate them from protein-bound material (l), rendered soluble by treatment of the extraction residue with trichloroacetic acid. Practically all the ADP and much of the ATP are found in the protein-bound fraction. The amount of bound ADP corresponds to the known actin content of mammalian muscle (2) and the well-established capacity of the fibrous form of this protein to bind ADP (2, 3). The only protein present in sufficient quantity to account for the bound ATP is myosin, and this only on the basis that one molecule of myosin binds several molecules of ATP. These data served as the basis for postulating that the bound ATP acts as the bridge between myosin and actin needed to account for the structure of resting muscle shown by the electron microscope. It] was also postu1 This work was supported AM 07273 from the National ritis and Metabolic diseases, IYational Science Folmdation partment, for an Undergradrlatc pation Program.
lated that this binding of ATP to myosin conforms to the requirements of an allosteric inhibitor of the ATPase action of myosin, by preventing the change in conformation about the active center necessary for the enzymic activity to be manifested (1). This type of inhibition can account for the failure to find direct evidence for the breakdown of ATP in cont’racting muscle (4, 5), or indirect evidence in the form of lack of the equilibration of 32P between phosphocreatine and terminal P of ATP that would be expected to result from the resynthesis of ATP by the creatine kinase reaction (6, 8). The present experiments were undertaken with two objectives. One was to compare amphibian with mammalian muscle with respect to the quant’itative relations between free and bound ADP and ATP. The other was to explore the possibility that the ATP was present in three functional compartments, rather than two, of which one might undergo dephosphorylation in eontraction and resynthesis by the creatine kinase reaction. If such were the case, there was the possibility that a tracer experiment would show equilibration of the isotope between phosphocreatine and the terminal P of the
in part by grant Institllte of Arthand in part by a grant to this deResearch Partici591
592
;
SACKS
ATP in this compartment only. Data were obtained that can be interpreted in terms of three such functional compartments. The results of the tracer experiments, to be reported later, do not indicate any tendency toward the establishment of isotopic equilibrium between the phosphocreatine and the terminal P of the ATP present in any one of the three compartments. The data on frog muscle show the same qualitative distribution of the ATP and ADP as in mammalian muscle. Quantitatively, very much less ATP is protein-bound in the frog muscle. EXPERIMENTAL
ET
musculature of the hind legs was used, with particular precautions to remove bone, cartilage, and tendons. The frozen muscles were powdered in a tissue crusher chilled with dry ice and treated in the Waring Blendor with 10 volumes of 80% ethanol saturated with EDTA, chilled to -2O”, let stand with occasional shaking for 10 minutes, and centrifuged at -20”. The residue was extracted twice with 5 volumes each of the 80% ethanol, then three times with 65y0 ethanol containing 0.001 M EDTA, and finally with 10% trichloroacetic acid. The separate extracts were treated as in the previous work (1) to remove small amounts of proteinaceous material in the ethanolic extracts, and with ether t)o remove trichloroacetic acid. The P compounds were absorbed on columns of Dowex-I resin. Elution of the extracts from the bullfrog muscles was begun with 0.002 N HCl, which would have eluted any AMP present. None was found, as had been the situation previously with mammalian muscle. ADP was eluted with 0.01 N HCl, and ATP with 0.01 N HCl + 0.04 M KCl, as before. Measurements of ADP and ATP in the various fractions of the eluates were made in terms of absorbance at 260 rnp.
PROCEDURE
The experiments on cats were carried out as in the earlier work (1) by pouring the freezing mixture over one gastocnemius muscle 15 seconds after the commencement of tetanic contraction under load, then freezing the resting muscle. Bullfrogs (Rana catesbiana) rather than grass frogs (Rana pipiens) were used in order to have muscle samples from one animal comparable in quantity to those obtained from the cats. The bullfrogs were stored in the cold room, allowed to warm up slowly overnight to room temperature-these experiments were done during the summer monthsand decerebrated with the point of a scapel. After recovery from the shock of the operation, one sciatic plexus was exposed high in the body cavity, stimulating electrodes were placed, and the nerve was cut proximal to the electrodes. The animal was then placed in a trough longer than the extended length, the tetanizing current was started, and 15 seconds later a large quantity of the freezing mixture, enough to freeze the entire animal, was portred into the trough. The entire
RESULTS
Muscle
Cat,
resting
Cat,
tetanized
Frog,
resting
Frog,
tetanized
OF ATP
AND
I AND
ADP
IN
MUSCLES
ATP FL%+
0.37 (0.03-O. 58) 0.39 (0.15-1.40) 0.87 (0.48-l. 76) 0.71 (0.32-l. 75)
a Values are rmoles/g, mean and range. * Free: extracted by 80% ethanol. Dissociable: ethanol; in trichloroacetic acid extract.
DISCUSSION
The data on the distribution of ADP and ATP in the three extraction media are summarized in Table I. In one of the experiments on cats, and in two of those on bullfrogs, faint traces of ADP were found in the 80% ethanolic extracts. The content of both ADP and ATP is significantly lower in bullfrog muscle than in cat muscle. The actin content of bullfrog muscle is not known, but Dolp (9) has shown
TABLE DISTRIBUTION
AL.
Dissociable
1.33 (0.88-l .64) 1.16 (0.26-2.12) 2.04 (1.18-3.02) 1.89 (0.74-3.40)
extracted
Bound
2.93 (1.61-3.99) 2.57 (1.66-3.93) 0.84 (0.20-l. 52) 0.90 (0.21-1.40)
by 65y0 ethanol.
ADP,
bound
No. expts.
0.53 (0.30-0.57) 0.44 (0.28-0.56) 0.33 (0.25-0.40) 0.37 (0.26-0.42)
Bound:
not
13 13 8 8
extracted
by
FREE
AND
BOUND
ADP
that the molecular weight of the monomer is the same as that reported by others for material from mammalian sources. On this basis, the present data suggest that all the actin and all the ADP in bullfrog muscle, as in mammalian, are present as the complex of F-actin with ADP, and that the ADP content can be taken as a measure of t’he actin content. While the data in viva and in vitro with respect to the protein binding of ADP are in good agreement, the same does not hold for ATP. The determination with isolated myosin is complicated by the ATPase action which appears under these conditions. Two reports (10, 11) give values which extrapolate to a value of 1 mole/mole of myosin, a value far lower than indicated by the findings in viva. The average amount of bound ATP in the bullfrog muscles corresponds to 5 or 6 moles/mole of myosin, on the assumptions that the myosin content of the muscles of this species is not significantly different from the 10% reported for rabbit muscle (12) and that the molecular weight is of the same order as that generally given for mammalian muscle, about 600,000. The value in the cat muscle corresponds to at least twice this molar ratio. The finding that much of the ATP not firmIy bound to protein is not extractable b-J 80% ethanol in the presence of EDTA, a medium in which the Na and K salts of ATP are freely soluble, suggests that the fraction extracted by 65 % ethanol may also be protein-bound, in a form more readily dissociable than that which requires denaturation of the protein to render the ATP extractable. The amount of this fraction is so great, especially in the bullfrog muscle, that t,he possibility needs to be considered that this fraction also is bound to myosin, in some other way than that which is firmly bound. This fraction is listed in the table as dissociable. Tetanic contraction does not alter significantly the distribution of ATP between free, dissociable, and firmly bound fractions. This situation is compatible with either the sliding filament theory or the one previously proposed (13), that the contraction is initiated by the breaking of the bridges on the
AND
ATP
IN
MUSCLE
593
actin side. The sliding filament theory does not require that the mat’erial constituting t’he bridge separate from both proteins in the breaking of the bridge. In the version proposed by Davies (14), retention of one ATP per H-meromyosin to the protein is specifically called for during the breaking and making of the bridge. The amount of firmly bound ATP is sufficient to allow one ATP-myosin bridge in the frog muscle, and two or more in the cat muscle, to each of the six actin filaments that the electron micrographs show to surround each myosin filament. In the theory proposed (13) as an alternate to the sliding filament, the rupture of the bond between the ATP and actin that allows the actin to pass into the disordered state does not require any breaking of the bonds between ATP and myosin. The present data do not permit a choice between the two theories; it does need to be pointed out that neither has an obligatory requirement for conversion of the bound ATP to ADP. REFERENCES 1. SACKS, J., MURPHREE, S., AND BROWN, It., Arch. Biochem. f?iophys.113,97 (1966). 2. HASSELRACH, W., B&him. Biophys. Ada 26, 652 (1957). 3. MARTONOSI, A., GOUVEA, M. A., AND GERGELY J., ./. Hiok Chem. 236, 1707 (1960); BARANY, M., NAGY, B., FINKELMAN, F., AND CHRAMBACH, A., J. Biol. Chem. 236, 2917 (1961). 4. MOMMAERTS, W. F. H. M., -am. J. Physiol. lE2, 585 (1955). 5. CIRLSON, F. D., AND SIGER, A.,J. Gen. Physiol. 44, 33 (1960). 6. FLECKENSTEIN, A., JANKE, J., LECHNER, M., I\ND BAuER, (i., i’jtuger’s drch. 269, 246 (1954). 7. DIXON, G. J., BND SACKS, J., ilm. J. Physiol. 193, 129 (1958). 8. Sacks, J., .IND CLELAND, M., Am. J. Physiol. 198, 300 (1960). 9. DOLP, R. M., Arch. Hiochem. Biophys. 113, 20 (1966). 10. NANNINGA, L. B., AND MOMM.IERTS, w. F. H. M., Proc. AVatl. Acad. Sci. U.S. 46, 1155 (1960). 11. MARTONOSI, A., AND MEYER, H., J. Biol. Chem. 239, 640 (1954). 12. HASSELBACH, w., AND SCHNEIDER, G., l?iothem. Z. 321, 462 (1951). 13. SACKS, J., Perspectives Biol. Med. 7,285 (1964). 14. D.~vIs, It. E., Nab-e 109, 1068 (1963).