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Physicochemical studies on denaturation of myosin—Adenosinetriphosphatase. II. Changes in chromatographic profile and optical rotation
ARCHIVES
OF
BIOCHEMISTRY
AND
Physicochemical
BIOPHYSICS
45-51
99,
Studies
on Denaturation
Adenosinetriphosphatase.
Profile
Optical TAKAHASHI,
of Myosin-
II. Changes
Chromatographic
KOUI
(1962)
in
and
Rotation
TSUTOMU YASUI, YOSHIO AND YUJI TONOMURA’
HASHIMOTO
From the Animal Husbandry Department, Faculty of Agriculture, Research Institute for Catalysis, Hoklcaido University, Sapporo, Received
March
and the Japan
9, 1962
The inactivation of myosin A-ATPase proceeds according to the first-order law. The rate constants of the inactivation were proportional to the 1.3,0, and -3.3 powers of [H+], respectively, in the ranges of pH 5.260, 7.58.5, and 10.0-10.5. In these three ranges of pH, AH$ values were 31.6, 52.6, and 42.1 kcal./mole, respectively. During the incubation at 36°C. the 01component in chromatographic profiles on diethylaminoethylcellulose decreased, the p component increased, and finally the fi component decomposed. However, specific ATPase activities of both the a and the 6 components were not constant and decreased with time. Under three conditions (pH 7.0 and 3O”C., pH 5.7 and 2O”C., and pH 10.3 and 2O”C.), the change in optical rotatory dispersion with time was measured and compared with that of ATPase activity. The helical content decreased very slowly and only by a few per cent, even after ATPase activity disappeared.
Pelletier and Ouellet (2) have measured the rate of inactivation of myosin A-ATPase over a wide range of pH and temperature. Connel (3) has observed that the inactivation of cod myosin A occurs along with intermolecular aggregation. Recently, Johnson and Lowe (4) have conducted detailed physicochemical studies on the denaturation of myosin A. Brahms (5) and Ferry (6) have separately carried out chromatography of myosin A on a diethylaminoethylcellulose (DEAE-cellulose) column, and the former author has suggested that the ,8 peak of Brahms (5) may be transformable into the cy peak during the incubation. In addition, t’hrough the recent work of Doty (7) and Kauzmann (8), it is generally accepted that the main event in the denaturation of proteins is a change in the secondary and tert’iary structures of the polypeptide chains. Taking these facts into consideration,
INTRODUCTION
Many reports have already appeared on the denaturation of myosin A. A kinetic investigation on the heat inactivation of (ATPmyosin A-adenosinetriphosphatase ase) at pH 7.0 has been performed by the present authors (l), and a reaction scheme of the inactivation has been proposed based primarily on the protecting effect of inorganic pyrophosphate on the inactivation. We (1) have also observed that the reduced viscositly of myosin A solution increases and a water-soluble fragment is released accompanying the heat inactivation of ATPase. 1 This investigation was supported by Research Grant A-4233 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, and by grants from Ministry of Education of Japan to the Research Groups on “Structure and Function of Muscle Protein” and on “Denaturation of Muscle Structural Protein.” 45
46
TAKAHASHI
rates of inactivation of myosin A-ATPase have been determined in a wider range of pH and temperature than previously employed, and changes in the chromatographic profile and in the optical rot,atory dispersion of myosin A have been measured during the process of inactivation, and the following results have been obtained: (a) During the incubation the (Y peak is transformed into the p peak, and the specific ATPase activities of both components are not constant but decrease with time; and (b) in the heat inactivation at pH 7.0 and in the acid or alkaline inactivation at 2O”C., the excess right-handed helical content of myosin A decreases only by a few per cent, even when the ATPase activity completely disappears. MATERIALS
AND
METHODS
Myosin A was prepared from rabbit skeletal muscle according to the method described by Perry (9) with slight modifications (10). The protein solution was stored at 0°C. The disodium salt of adenosine triphosphate IATP) (Sigma Chemical Co.) was used throughnut these experiments. Its concentration was determined by a Hitachi spectrophotometer from nltraviolet absorption at 2600 A. h1ne milliliter of about 0.5% protein solution m 0 5 J4 KC1 was added to 1 ml. of 0.5 M KC1 solution containing 100 mM buffer (Tris-maleate-NaOH arm glycine-NaOH were used as buffers in the pH ranges of 5.2 to 8.5 and above 8.5, respectively) which was already equilibrated to the required t,emperature. At measured intervals of time, 0.2 ml. samples were withdrawn and pipetted into 2 ml. of cold solubion of 0.5 M KCl, 50 mM Trisma!eate (pH 7.0), and 10 mM CaCl* , unless otherwise stated. The ATPase activity of the resulting mixture was assayed at 21°C. by measuring the amount of orthophosphate liberated by the method of Martin and Doty (II), since inactivation of ATPase at various conditions of pH and temperature was always found to be irreversible. Diethylaminoethylcellulose was prepared by the method of Peterson and Sober (12). The chromatography of myosin A on DEAE-cellulose was in general carried out, as described by Perry and Zydwo (13): A myosin A solution of about 0.57” was equilibrated by dialysis against 0.25 M KC1 and 10 mM Tris-HCl buffer (pH 7.8), centrifuged at 5 X IO4 g. for 1 hr., and then run into a column (2.3 X 20 cm.) of DEAE-cellulose equilihrated against the same buffer. After the fraction
ET AL. which was not adsorbed under these conditions has passed through the column, an ascending (pH 7.8) gradient to 1 M KC1 and 10 mM Tris-HCl was applied. Ultraviolet absorption at 2800 A. and ATPase activity were measured in the effluents. The optical rotation of 0.5 M KC1 solution which contained 50 mM buffer and 5 mg./ml. myosin A was measured by means of a model 200 S-80 photoelectric spectropolarimeter with an oscillating polarizer prism (0. C. Rudolph and Sons) in the range of 3500 to 5890 A. All the results were expressed in terms of the equation of Moffitt and Yang (14) : [&I
= 3[Rl n2 +
2
= __uow
~ boM
x2 - x02+ (x2 - xr#
Here the residue rotation [RI is [e] X mean residue weight/loo, X is the wavelength at which the observation is made, 71 is the refractive index of the solvent, and a~ and ba are constants derived by fitting the data to the equation. The adjustable parameter, X0 , was taken as constant and equal to 2140 A. Excess right-handed a-helical content was calculated by dividing the bo term by -580, as suggested by Doty (7) Determination of nitrogen content by the micro-Kjeldahl method was used as primary standard in determining protein concentration, a value of 16.2y0 being assumed for the nitrogen content of myosin A. Ultraviolet absorption at 2800 A. was also employed. RESULTS
KINETIC
PROPERTIES
As shown in Fig. 1, the heat inactivation of myosin A-ATPase at pH 7.0 proceeded according to the first-order law, and the rate constant, lco, of inactivation was independent of protein concentration. The enthalpy of activation, AH& of the inactivation process was 56.2 Kcal./mole at pH 7.0, this value agreeing with the reports of Pelletier and Ouellet (2) and our previous paper (15). The pH dependency of ATPase activity was not affected even by the reduction of the activity by heat to 50 % of the original. In Fig. 2 are shown the pH dependences of Ic, measured in the pH range from 5.2 to 10.5 at 10, 15, 20, and 25°C. As clearly seen in this figure, the dependence of k, upon pH was almost t’he same at these four different temperatures. As already reported (2, 15),
DENATURATION
OF MYOSIN-ATPASE.
12, was independent of [H+] in the range of pH of 7.5-8.5. Below pH 6.0, k, was proportional to [H+]*.3, and above pH 10.0 k, was proportional to l/[H+]“.“. The latter order is smaller than the 4.5 reported by Pelletier and Ouellet (2). The orders of the dependence of k, upon [H+] in these three ranges of pH were constant and independent of temperature. Since these experiments were carried out during a relatively long period, it seemed to be undesirable to determine AHI from these figures. Therefore, Ic, was determined for a fresh myosin A preparation at pH’s 5.Fj, 8.0, and 10.3. The results are shown in Fig. 3, and AHJ. values were calculated to be 31.6, 52.6, and 42.1 kcal./ mole, respectively. CHROMATOGRAPIIIC
PROFILE
A typical example of the change in chromatographic profile of myosin A during incubation at 36°C. is presented in Fig. 4, A-D. The original myosin A resolved into two components, OLand p, as designated by Brahms (5). The p component occupied, however, only about 5 % or at most 8% of the total area (A). During the incubation a series of changes appeared on chromato-
II
FIG. 2. Inactivation constant, ko (in set .-I), as a function of pH at different temperatures. For measurements of ko , myosin A was incubat)ed in 0.5 M KC1 and 50 mM buffer at 25°C. (O), 20°C. (X), 15°C. (A), or 10°C. (0). ATPase assay as described in Fig. 1.
* I , 3.3
35
34
36
‘~WY FIG. 3. Dependence on temperature of ko of myosin A-ATPase at different pH’s. Myosin A was incubated in 0.5 M KC1 and 50 mM buffer and at pH 5.5 (0)) pH 8.0 (X), or pH 10.3 ( l ) ATPase assay as described in Fig. 1.
lncubotion
Time (HOurS)
FIG. 1. Decrease in ATPase activity of myosin A as a function of time during storage in 0.5 M KC1 and 50 mM Tris-maleate (pH 7.0) at different temperatures. 0, 35°C.; X, 30°C.; A, 25”C., 0, 20°C.; Q , 15°C. ATPase activity was assayed in 0.5 M KCl, 50 mM Tris-maleate (pH 7.0), 1OmM CsClz and 1 mM ATP at 21°C. and expressed as logarithms of percentage of the original activity.
graphic profiles : The (Ypeak was transformed into the p peak (B, C) and finally the p peak decomposed (D). Figure 4 also contains the total ATPase activity of each fraction. The curve obt,ained by plotting the ATPase activity coincided with a plot of absorption at 2800 A. This indicates that both the (Y and the ,~3components have ATPase activity. In Fig. 5 are presented the changes during the incubation at 36°C. in the amounts of the cy and the P components together with
48
TAKAHASHI
ET AL.
(0) a6
50
100
I50 Volume of Eldml)
Volume of Elute(ml)
Volume of Elutehnl)
Volume of Elute (ml)
FIG. 4. Chromatographic profile of myosin A on diethylaminoethylcellulose and ATPase activity of effluent. Myosin A was incubated in 0.25 M KC1 and 10 mM Tris-NC1 (pH 7.8) at 36°C. for 0 hr. (A); 0.5 hr. (B); 1 hr. (C) ; and 8 hr. (D). Ten milliliters of protein solution (El em.2.58 at 280 mN) in 0.25 M KC1 and 10 mM Tris-HCl (pH 7.8) was applied to a column (2.3 X 20 cm). Elution by gradient to 1 M KC1 and 10 mM Tris-HCl (pH 7.8) was applied at the arrow. Flow rate was 60 ml./hr. Six-milliliter fractions were collected. 0,E. 980mp ; 0, ATPase activity. After dialysis of effluent against 0.5 M KCl, 10 mM CaClz solution (pH 7.0) at O”C., ATPase activity was assayed at 21°C. by adding 1 mM ATP.
specific ATPase activities. During the incubation, specific ATPase activities of both the 01and the p components decreased with time, and the ATPase activity of the apparently same component on the chromatograms varied extremely. In addition, there were no detectable differences between the (Y and the p components in their pHATPase activity curves. their
It
must
be added
that
the rate
of the
transformation in chromatographic profiles described above varied markedly with myosin A preparations used. Our investigation on chromatographic behavior of myosin A was carried out from June 1960 to December 1961. In the case of myosin A prepared in June to August, the p component increased to only 19.3% of the total by incubation at 36°C. for 30 min.; but in t,he case of myosin A prepared in October
DENATURATION
OF MYOSIN-ATPASE.
to December, the p component reached 41.2% under the same conditions. The reason for this marked difference is not yet clear, but the difference might be due to the seasonal change in the microstructure of the myosin A molecule. Although t’he time-course in changes in chromatographic profiles varied, the rate of inactivation of ATPase activity did not fluctuat’e with season. OPTICAL
ROTATORY
49
II
*
100
(0,O.A) ‘\
(aA)
DISPERSION
After change of pH of the myosin il solution from 7.0 to 5.7 at 2O”C., the ---a0 term increased and the -bo term decreased gradually. Figure 6 indicates the time course of change of excess right-handed a-helical content’ estimated from the bo term by Doty’s method (7), together with the decrease in ATPase activity at pH 5.7 and 20°C. The changes with time in helical content’ and ATPase activity at pH 10.3 and 20°C. are also included in this figure. As previously reported (16-18)) helical content of myosin A at pH 7.0 and at room temperature was about 60%, though it loot (O.A) t 01
\
\ \ ‘A.
0
2
4 Incubation
g m
‘. .‘a. -S -5s -A-,-
6
6
” to 10
Time (Hours)
FIG. 6. Time-courses of changes in helical content and in ATPase activity: 5 mg./ml. of myosin A was incubated in 0.5 M KC1 and 50 mM buffer and at 20°C. Helical content was measured from optical rotatory dispersion curve of myosin A at pH 5.7 (O), 10.3 (A), or pH 7.0 (O).Decreasein ATPase activity at pH 5.7 ( l ) or pH 10.3 (A) was assayed as described in Fig. 1.
-4 (aA)
0
0.5 Incubation
;
(J-p
Time (Hours)
FIG. 5. Time-courses of changes in amounts of a(O) and /3(A) components and in specific ATPase
activities of a(O) tions of incubation assay as described
and p(A) components. Condiof myosin A and of ATPase in Fig. 3.
varied to some extent from one preparation to another. Both at pH’s 5.7 and 10.3 helical content decreased very gradually with time. Ten hours after changing the pH from 7.0 to 5.7 or 10.3, helical content decreased only by about 5 %, while ATPase activity disappeared almost completely. The optical rotatory dispersion of myosin ,4 changed with time at pH 7.0 and 30°C. very similarly to those in the acid or the alkaline media; i.e., the --a0 term increased and the --b. term decreased gradually. In P’ig. 7 are plotted the time-course of changes in helical content and ATPase activit,y at pH 7.0 and 30°C. The helical content decreased by only a few per cent aft,er 10 hr. incubation at pH 7.0 and 3O”C., although ATPase activity almost completely disappeared. Connel (3) observed a slight change in optical rotation of cod myosin during incubation at 23”C., but the opt’ical rotatory power was determined only at 5890 A, and no time study of the change was performed.
TAKAHASHI
f
I
8
2
4
6
6
lncubotion Time hours)
FIG. 7. Time-courses of changes in helical cont,ent and in ATPase activity: 5 mg./ml. of myosin A was incubated in 0.5 M KC1 and 50 m&f Trismaleate (pH 7.0) and at 30°C. Helical content was measuredfrom optical rotatory dispersion of myosin A solution at 30°C. (0) or 20°C. (0). ATPase (e) assay as described in Fig. 1. DISCUSSION
In the chromatographic profile of myosin A the a: peak is transformed into the /3 peak during incubation at 36”C., but specific ATPase activities of these components were not constant, but decreasing with time. Furthermore, in all three media investigated the excess right-handed a-helical content, estimated from t!he bo term following Doty (7), decreased only by a few per cent, even when ATPase activity disappeared. These facts may be interpreted by Lhe following alternatives: (a) the structure of the active site of ATPase is nonhelical and (b) the structure is helical and there is a localized disorganization in the denaturation media. Recently, the present authors (1) and Johnson and Lowe (4) have observed that the viscosity of myosin A increases and a water-soluble fragment appears accompanying the denaturation of myosin A. Furthermore, Johnson and Lowe (4) have observed changes in sedimentation rate and in partial specific volume of myosin A during the denaturation process. From these results
ET AL.
Johnson and Lowe (4) have concluded that the denaturation is due to an alteration of the tertiary structure in the myosin A molecule together with intermolecular aggregations. On the other hand, one of the present authors (10, 17, 18) has recently made the following observations: (a) The addition of inorganic pyrophosphate, a competitive inhibitor of ATPase, changes the optical rotatory dispersion of myosin A. (6) Imm~iately after the addition of 10% dioxane, ATPase activity is enhanced to about 150 % of the original; then it decreases gradually and finally disappears. In this case the helical content first increases by a few per cent and thereafter decreases gradually. Even when ATPase activity completely disappeared, the helical content is lower than the original by only a few per cent. (c) On the other hand, the helical content of the alkaline-denatured myosin A and the pyrophosphat~-myosill A complex does not change on adding dioxane. When pyrophosphate is added into the myosin A solution at various times after the addition of dioxane, t#heshift in helical content caused by dioxane is not observed any more, and the content remains constant over the period of measurements. These and the present results suggest, that a remarkable change in ATPase activity is induced by a change in helical content of a few per cent, though the possibility that a change in the conformation of nonhelical part of the molecule induces a remarkable change in ATPase activity cannot of course be excluded. The chromatographic investigations mentioned above also suggest that a slight change in the secondary and tbe tertiary structures induces inactivation of ATPase and the change from the LY to the 8 component occurs independently of the ATPase inactivation. A slight change in optical rotation during denaturation of chymotrypsin has been reported by Schwert (19) and by Brandts and Lumry (20). ACKNOWLEDGMENTS The aut,hors are indebted to Prof. M. F. Morales of California University for his helpful suggestions on ~hroma~ographic investigation on den~turatiop of myosin A.
DENATURATIOK
OF MYOSIN-ATPASE.
REFERENCES 1. YASUI, T., HASHIMOTO, Y., AND TONOMURA, Y., Arch. Biochem. Biophys. 87, 55 (1960). 2. PELLETIER, G. E., AND OUELLET, L., Can. J. Chem. 39, 265 (1961). 3. CONNEL, J. J., Rio&em. J. 75, 530 (1960). 4. JOHNSON, P., AND LOWE, A. J., Biochim. et Biophys. Acta 53, 343 (1961). 5. BRAHMS, J., J. Am. Chem. Sot. 81, 4997 (1959). 6. PERRY, S. V., Biochem. J. 74, 97 (1960). 7. DOTY, P., Proc. Intern. Symposium on Macromolecular Chem., Prague, 196Y, p. 5. (Pergamon Press, London, 1957). 8. KAUZMANN, W., Advances in Protein, Chem, 14, 1 (1957). 9. PERRY, S. V., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 582. Academic Press, New York, 1955. 10. TONOMURA, Y., TOKURA, S., SEKIYA, K., AND IMAMURA, K., Arch. Biochem. Biophys. 95, 229 (1961).
II
51
11. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 12. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 71, 751 (1956). 13. PERRY, S. V., AND ZYDWO, M., Biochem. J. 71, 220 (1959). 14. MOFFITT, W., AND YANG, J. T., Proc. Natl. Acad. Sci. U. S. 42, 596 (1959). 15. YASUI, T., FUKAZAWA, T., HASHIMOTO, Y., KITAGAWA, S., AND SASAKI, A. T., J. Biothem. (Tokyo) 45, 717 (1958). 16. COHEN, C., AND SZENT-GYORGYI, A. G., J. Am. Chem. Sot. 79, 248 (1957). 17. TONOMURA, Y., TOKURA, S., AND SEKIYA, K., J. Biol. Chem. 237, 1074 (1962). 18. TOXOMURA, Y., SEKIYA, K., AND IMAMURA, K., Biochim. et Biophys. Acta, in press. 19. SCHWERT, G. W., cited in KAUZMANN, W., J. Cellular Camp. Physiol. 47, Suppl. 1, 126 (1956). 20. BRANDTS, J., AND LUMRY, R., J. Am. Chem. Sot. 83, 4290 (1961).
OF
BIOCHEMISTRY
AND
Physicochemical
BIOPHYSICS
45-51
99,
Studies
on Denaturation
Adenosinetriphosphatase.
Profile
Optical TAKAHASHI,
of Myosin-
II. Changes
Chromatographic
KOUI
(1962)
in
and
Rotation
TSUTOMU YASUI, YOSHIO AND YUJI TONOMURA’
HASHIMOTO
From the Animal Husbandry Department, Faculty of Agriculture, Research Institute for Catalysis, Hoklcaido University, Sapporo, Received
March
and the Japan
9, 1962
The inactivation of myosin A-ATPase proceeds according to the first-order law. The rate constants of the inactivation were proportional to the 1.3,0, and -3.3 powers of [H+], respectively, in the ranges of pH 5.260, 7.58.5, and 10.0-10.5. In these three ranges of pH, AH$ values were 31.6, 52.6, and 42.1 kcal./mole, respectively. During the incubation at 36°C. the 01component in chromatographic profiles on diethylaminoethylcellulose decreased, the p component increased, and finally the fi component decomposed. However, specific ATPase activities of both the a and the 6 components were not constant and decreased with time. Under three conditions (pH 7.0 and 3O”C., pH 5.7 and 2O”C., and pH 10.3 and 2O”C.), the change in optical rotatory dispersion with time was measured and compared with that of ATPase activity. The helical content decreased very slowly and only by a few per cent, even after ATPase activity disappeared.
Pelletier and Ouellet (2) have measured the rate of inactivation of myosin A-ATPase over a wide range of pH and temperature. Connel (3) has observed that the inactivation of cod myosin A occurs along with intermolecular aggregation. Recently, Johnson and Lowe (4) have conducted detailed physicochemical studies on the denaturation of myosin A. Brahms (5) and Ferry (6) have separately carried out chromatography of myosin A on a diethylaminoethylcellulose (DEAE-cellulose) column, and the former author has suggested that the ,8 peak of Brahms (5) may be transformable into the cy peak during the incubation. In addition, t’hrough the recent work of Doty (7) and Kauzmann (8), it is generally accepted that the main event in the denaturation of proteins is a change in the secondary and tert’iary structures of the polypeptide chains. Taking these facts into consideration,
INTRODUCTION
Many reports have already appeared on the denaturation of myosin A. A kinetic investigation on the heat inactivation of (ATPmyosin A-adenosinetriphosphatase ase) at pH 7.0 has been performed by the present authors (l), and a reaction scheme of the inactivation has been proposed based primarily on the protecting effect of inorganic pyrophosphate on the inactivation. We (1) have also observed that the reduced viscositly of myosin A solution increases and a water-soluble fragment is released accompanying the heat inactivation of ATPase. 1 This investigation was supported by Research Grant A-4233 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, and by grants from Ministry of Education of Japan to the Research Groups on “Structure and Function of Muscle Protein” and on “Denaturation of Muscle Structural Protein.” 45
46
TAKAHASHI
rates of inactivation of myosin A-ATPase have been determined in a wider range of pH and temperature than previously employed, and changes in the chromatographic profile and in the optical rot,atory dispersion of myosin A have been measured during the process of inactivation, and the following results have been obtained: (a) During the incubation the (Y peak is transformed into the p peak, and the specific ATPase activities of both components are not constant but decrease with time; and (b) in the heat inactivation at pH 7.0 and in the acid or alkaline inactivation at 2O”C., the excess right-handed helical content of myosin A decreases only by a few per cent, even when the ATPase activity completely disappears. MATERIALS
AND
METHODS
Myosin A was prepared from rabbit skeletal muscle according to the method described by Perry (9) with slight modifications (10). The protein solution was stored at 0°C. The disodium salt of adenosine triphosphate IATP) (Sigma Chemical Co.) was used throughnut these experiments. Its concentration was determined by a Hitachi spectrophotometer from nltraviolet absorption at 2600 A. h1ne milliliter of about 0.5% protein solution m 0 5 J4 KC1 was added to 1 ml. of 0.5 M KC1 solution containing 100 mM buffer (Tris-maleate-NaOH arm glycine-NaOH were used as buffers in the pH ranges of 5.2 to 8.5 and above 8.5, respectively) which was already equilibrated to the required t,emperature. At measured intervals of time, 0.2 ml. samples were withdrawn and pipetted into 2 ml. of cold solubion of 0.5 M KCl, 50 mM Trisma!eate (pH 7.0), and 10 mM CaCl* , unless otherwise stated. The ATPase activity of the resulting mixture was assayed at 21°C. by measuring the amount of orthophosphate liberated by the method of Martin and Doty (II), since inactivation of ATPase at various conditions of pH and temperature was always found to be irreversible. Diethylaminoethylcellulose was prepared by the method of Peterson and Sober (12). The chromatography of myosin A on DEAE-cellulose was in general carried out, as described by Perry and Zydwo (13): A myosin A solution of about 0.57” was equilibrated by dialysis against 0.25 M KC1 and 10 mM Tris-HCl buffer (pH 7.8), centrifuged at 5 X IO4 g. for 1 hr., and then run into a column (2.3 X 20 cm.) of DEAE-cellulose equilihrated against the same buffer. After the fraction
ET AL. which was not adsorbed under these conditions has passed through the column, an ascending (pH 7.8) gradient to 1 M KC1 and 10 mM Tris-HCl was applied. Ultraviolet absorption at 2800 A. and ATPase activity were measured in the effluents. The optical rotation of 0.5 M KC1 solution which contained 50 mM buffer and 5 mg./ml. myosin A was measured by means of a model 200 S-80 photoelectric spectropolarimeter with an oscillating polarizer prism (0. C. Rudolph and Sons) in the range of 3500 to 5890 A. All the results were expressed in terms of the equation of Moffitt and Yang (14) : [&I
= 3[Rl n2 +
2
= __uow
~ boM
x2 - x02+ (x2 - xr#
Here the residue rotation [RI is [e] X mean residue weight/loo, X is the wavelength at which the observation is made, 71 is the refractive index of the solvent, and a~ and ba are constants derived by fitting the data to the equation. The adjustable parameter, X0 , was taken as constant and equal to 2140 A. Excess right-handed a-helical content was calculated by dividing the bo term by -580, as suggested by Doty (7) Determination of nitrogen content by the micro-Kjeldahl method was used as primary standard in determining protein concentration, a value of 16.2y0 being assumed for the nitrogen content of myosin A. Ultraviolet absorption at 2800 A. was also employed. RESULTS
KINETIC
PROPERTIES
As shown in Fig. 1, the heat inactivation of myosin A-ATPase at pH 7.0 proceeded according to the first-order law, and the rate constant, lco, of inactivation was independent of protein concentration. The enthalpy of activation, AH& of the inactivation process was 56.2 Kcal./mole at pH 7.0, this value agreeing with the reports of Pelletier and Ouellet (2) and our previous paper (15). The pH dependency of ATPase activity was not affected even by the reduction of the activity by heat to 50 % of the original. In Fig. 2 are shown the pH dependences of Ic, measured in the pH range from 5.2 to 10.5 at 10, 15, 20, and 25°C. As clearly seen in this figure, the dependence of k, upon pH was almost t’he same at these four different temperatures. As already reported (2, 15),
DENATURATION
OF MYOSIN-ATPASE.
12, was independent of [H+] in the range of pH of 7.5-8.5. Below pH 6.0, k, was proportional to [H+]*.3, and above pH 10.0 k, was proportional to l/[H+]“.“. The latter order is smaller than the 4.5 reported by Pelletier and Ouellet (2). The orders of the dependence of k, upon [H+] in these three ranges of pH were constant and independent of temperature. Since these experiments were carried out during a relatively long period, it seemed to be undesirable to determine AHI from these figures. Therefore, Ic, was determined for a fresh myosin A preparation at pH’s 5.Fj, 8.0, and 10.3. The results are shown in Fig. 3, and AHJ. values were calculated to be 31.6, 52.6, and 42.1 kcal./ mole, respectively. CHROMATOGRAPIIIC
PROFILE
A typical example of the change in chromatographic profile of myosin A during incubation at 36°C. is presented in Fig. 4, A-D. The original myosin A resolved into two components, OLand p, as designated by Brahms (5). The p component occupied, however, only about 5 % or at most 8% of the total area (A). During the incubation a series of changes appeared on chromato-
II
FIG. 2. Inactivation constant, ko (in set .-I), as a function of pH at different temperatures. For measurements of ko , myosin A was incubat)ed in 0.5 M KC1 and 50 mM buffer at 25°C. (O), 20°C. (X), 15°C. (A), or 10°C. (0). ATPase assay as described in Fig. 1.
* I , 3.3
35
34
36
‘~WY FIG. 3. Dependence on temperature of ko of myosin A-ATPase at different pH’s. Myosin A was incubated in 0.5 M KC1 and 50 mM buffer and at pH 5.5 (0)) pH 8.0 (X), or pH 10.3 ( l ) ATPase assay as described in Fig. 1.
lncubotion
Time (HOurS)
FIG. 1. Decrease in ATPase activity of myosin A as a function of time during storage in 0.5 M KC1 and 50 mM Tris-maleate (pH 7.0) at different temperatures. 0, 35°C.; X, 30°C.; A, 25”C., 0, 20°C.; Q , 15°C. ATPase activity was assayed in 0.5 M KCl, 50 mM Tris-maleate (pH 7.0), 1OmM CsClz and 1 mM ATP at 21°C. and expressed as logarithms of percentage of the original activity.
graphic profiles : The (Ypeak was transformed into the p peak (B, C) and finally the p peak decomposed (D). Figure 4 also contains the total ATPase activity of each fraction. The curve obt,ained by plotting the ATPase activity coincided with a plot of absorption at 2800 A. This indicates that both the (Y and the ,~3components have ATPase activity. In Fig. 5 are presented the changes during the incubation at 36°C. in the amounts of the cy and the P components together with
48
TAKAHASHI
ET AL.
(0) a6
50
100
I50 Volume of Eldml)
Volume of Elute(ml)
Volume of Elutehnl)
Volume of Elute (ml)
FIG. 4. Chromatographic profile of myosin A on diethylaminoethylcellulose and ATPase activity of effluent. Myosin A was incubated in 0.25 M KC1 and 10 mM Tris-NC1 (pH 7.8) at 36°C. for 0 hr. (A); 0.5 hr. (B); 1 hr. (C) ; and 8 hr. (D). Ten milliliters of protein solution (El em.2.58 at 280 mN) in 0.25 M KC1 and 10 mM Tris-HCl (pH 7.8) was applied to a column (2.3 X 20 cm). Elution by gradient to 1 M KC1 and 10 mM Tris-HCl (pH 7.8) was applied at the arrow. Flow rate was 60 ml./hr. Six-milliliter fractions were collected. 0,E. 980mp ; 0, ATPase activity. After dialysis of effluent against 0.5 M KCl, 10 mM CaClz solution (pH 7.0) at O”C., ATPase activity was assayed at 21°C. by adding 1 mM ATP.
specific ATPase activities. During the incubation, specific ATPase activities of both the 01and the p components decreased with time, and the ATPase activity of the apparently same component on the chromatograms varied extremely. In addition, there were no detectable differences between the (Y and the p components in their pHATPase activity curves. their
It
must
be added
that
the rate
of the
transformation in chromatographic profiles described above varied markedly with myosin A preparations used. Our investigation on chromatographic behavior of myosin A was carried out from June 1960 to December 1961. In the case of myosin A prepared in June to August, the p component increased to only 19.3% of the total by incubation at 36°C. for 30 min.; but in t,he case of myosin A prepared in October
DENATURATION
OF MYOSIN-ATPASE.
to December, the p component reached 41.2% under the same conditions. The reason for this marked difference is not yet clear, but the difference might be due to the seasonal change in the microstructure of the myosin A molecule. Although t’he time-course in changes in chromatographic profiles varied, the rate of inactivation of ATPase activity did not fluctuat’e with season. OPTICAL
ROTATORY
49
II
*
100
(0,O.A) ‘\
(aA)
DISPERSION
After change of pH of the myosin il solution from 7.0 to 5.7 at 2O”C., the ---a0 term increased and the -bo term decreased gradually. Figure 6 indicates the time course of change of excess right-handed a-helical content’ estimated from the bo term by Doty’s method (7), together with the decrease in ATPase activity at pH 5.7 and 20°C. The changes with time in helical content’ and ATPase activity at pH 10.3 and 20°C. are also included in this figure. As previously reported (16-18)) helical content of myosin A at pH 7.0 and at room temperature was about 60%, though it loot (O.A) t 01
\
\ \ ‘A.
0
2
4 Incubation
g m
‘. .‘a. -S -5s -A-,-
6
6
” to 10
Time (Hours)
FIG. 6. Time-courses of changes in helical content and in ATPase activity: 5 mg./ml. of myosin A was incubated in 0.5 M KC1 and 50 mM buffer and at 20°C. Helical content was measured from optical rotatory dispersion curve of myosin A at pH 5.7 (O), 10.3 (A), or pH 7.0 (O).Decreasein ATPase activity at pH 5.7 ( l ) or pH 10.3 (A) was assayed as described in Fig. 1.
-4 (aA)
0
0.5 Incubation
;
(J-p
Time (Hours)
FIG. 5. Time-courses of changes in amounts of a(O) and /3(A) components and in specific ATPase
activities of a(O) tions of incubation assay as described
and p(A) components. Condiof myosin A and of ATPase in Fig. 3.
varied to some extent from one preparation to another. Both at pH’s 5.7 and 10.3 helical content decreased very gradually with time. Ten hours after changing the pH from 7.0 to 5.7 or 10.3, helical content decreased only by about 5 %, while ATPase activity disappeared almost completely. The optical rotatory dispersion of myosin ,4 changed with time at pH 7.0 and 30°C. very similarly to those in the acid or the alkaline media; i.e., the --a0 term increased and the --b. term decreased gradually. In P’ig. 7 are plotted the time-course of changes in helical content and ATPase activit,y at pH 7.0 and 30°C. The helical content decreased by only a few per cent aft,er 10 hr. incubation at pH 7.0 and 3O”C., although ATPase activity almost completely disappeared. Connel (3) observed a slight change in optical rotation of cod myosin during incubation at 23”C., but the opt’ical rotatory power was determined only at 5890 A, and no time study of the change was performed.
TAKAHASHI
f
I
8
2
4
6
6
lncubotion Time hours)
FIG. 7. Time-courses of changes in helical cont,ent and in ATPase activity: 5 mg./ml. of myosin A was incubated in 0.5 M KC1 and 50 m&f Trismaleate (pH 7.0) and at 30°C. Helical content was measuredfrom optical rotatory dispersion of myosin A solution at 30°C. (0) or 20°C. (0). ATPase (e) assay as described in Fig. 1. DISCUSSION
In the chromatographic profile of myosin A the a: peak is transformed into the /3 peak during incubation at 36”C., but specific ATPase activities of these components were not constant, but decreasing with time. Furthermore, in all three media investigated the excess right-handed a-helical content, estimated from t!he bo term following Doty (7), decreased only by a few per cent, even when ATPase activity disappeared. These facts may be interpreted by Lhe following alternatives: (a) the structure of the active site of ATPase is nonhelical and (b) the structure is helical and there is a localized disorganization in the denaturation media. Recently, the present authors (1) and Johnson and Lowe (4) have observed that the viscosity of myosin A increases and a water-soluble fragment appears accompanying the denaturation of myosin A. Furthermore, Johnson and Lowe (4) have observed changes in sedimentation rate and in partial specific volume of myosin A during the denaturation process. From these results
ET AL.
Johnson and Lowe (4) have concluded that the denaturation is due to an alteration of the tertiary structure in the myosin A molecule together with intermolecular aggregations. On the other hand, one of the present authors (10, 17, 18) has recently made the following observations: (a) The addition of inorganic pyrophosphate, a competitive inhibitor of ATPase, changes the optical rotatory dispersion of myosin A. (6) Imm~iately after the addition of 10% dioxane, ATPase activity is enhanced to about 150 % of the original; then it decreases gradually and finally disappears. In this case the helical content first increases by a few per cent and thereafter decreases gradually. Even when ATPase activity completely disappeared, the helical content is lower than the original by only a few per cent. (c) On the other hand, the helical content of the alkaline-denatured myosin A and the pyrophosphat~-myosill A complex does not change on adding dioxane. When pyrophosphate is added into the myosin A solution at various times after the addition of dioxane, t#heshift in helical content caused by dioxane is not observed any more, and the content remains constant over the period of measurements. These and the present results suggest, that a remarkable change in ATPase activity is induced by a change in helical content of a few per cent, though the possibility that a change in the conformation of nonhelical part of the molecule induces a remarkable change in ATPase activity cannot of course be excluded. The chromatographic investigations mentioned above also suggest that a slight change in the secondary and tbe tertiary structures induces inactivation of ATPase and the change from the LY to the 8 component occurs independently of the ATPase inactivation. A slight change in optical rotation during denaturation of chymotrypsin has been reported by Schwert (19) and by Brandts and Lumry (20). ACKNOWLEDGMENTS The aut,hors are indebted to Prof. M. F. Morales of California University for his helpful suggestions on ~hroma~ographic investigation on den~turatiop of myosin A.
DENATURATIOK
OF MYOSIN-ATPASE.
REFERENCES 1. YASUI, T., HASHIMOTO, Y., AND TONOMURA, Y., Arch. Biochem. Biophys. 87, 55 (1960). 2. PELLETIER, G. E., AND OUELLET, L., Can. J. Chem. 39, 265 (1961). 3. CONNEL, J. J., Rio&em. J. 75, 530 (1960). 4. JOHNSON, P., AND LOWE, A. J., Biochim. et Biophys. Acta 53, 343 (1961). 5. BRAHMS, J., J. Am. Chem. Sot. 81, 4997 (1959). 6. PERRY, S. V., Biochem. J. 74, 97 (1960). 7. DOTY, P., Proc. Intern. Symposium on Macromolecular Chem., Prague, 196Y, p. 5. (Pergamon Press, London, 1957). 8. KAUZMANN, W., Advances in Protein, Chem, 14, 1 (1957). 9. PERRY, S. V., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 582. Academic Press, New York, 1955. 10. TONOMURA, Y., TOKURA, S., SEKIYA, K., AND IMAMURA, K., Arch. Biochem. Biophys. 95, 229 (1961).
II
51
11. MARTIN, J. B., AND DOTY, D. M., Anal. Chem. 21, 965 (1949). 12. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 71, 751 (1956). 13. PERRY, S. V., AND ZYDWO, M., Biochem. J. 71, 220 (1959). 14. MOFFITT, W., AND YANG, J. T., Proc. Natl. Acad. Sci. U. S. 42, 596 (1959). 15. YASUI, T., FUKAZAWA, T., HASHIMOTO, Y., KITAGAWA, S., AND SASAKI, A. T., J. Biothem. (Tokyo) 45, 717 (1958). 16. COHEN, C., AND SZENT-GYORGYI, A. G., J. Am. Chem. Sot. 79, 248 (1957). 17. TONOMURA, Y., TOKURA, S., AND SEKIYA, K., J. Biol. Chem. 237, 1074 (1962). 18. TOXOMURA, Y., SEKIYA, K., AND IMAMURA, K., Biochim. et Biophys. Acta, in press. 19. SCHWERT, G. W., cited in KAUZMANN, W., J. Cellular Camp. Physiol. 47, Suppl. 1, 126 (1956). 20. BRANDTS, J., AND LUMRY, R., J. Am. Chem. Sot. 83, 4290 (1961).