THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 2, Issue of January 25, pp. 219-224, 1970

PTinted in U.S.A.

Differences between Slow and Fast Muscle Myosin

ADENOSINE TRIPHOSPHATASE ACTIVITY AND RELEASE OF ASSOCIATED PROTEINS BY p-CHLOROMERCURIPHENYLSULFONATE

(Received for publication, January 3, 1969)

FREDERICK J. SAMAHA, LLOYD GUTH, AND R. WAYNE ALBERS

From the Laboratory of Neurochemistry and Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological Diseases and Xtroke, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

The differences between slow and fast muscle myosin adenosine triphosphatase activity of the cat are described with reference to relative specific activities, p-chloromercuri- phenylsulfonate activation, and alkaline and acid stability.

The treatment of fast muscle myosin with 8 moles of p- chloromercnriphenylsulfonate per lo5 g of myosin results in the release of two electrophoretically distinct proteins and loss of myosin ATPase activity.

There appears to be an inverse relationship between the degree of ATPase activity lost and the amount of closely associated proteins released from fast muscle myosin. Similar treatment of slow muscle myosin releases no proteins and causes no loss of ATPase activity.

Recent studies on rabbit muscle have shown that myosins ob- tained from white (fast) muscle and from red (slow) muscle have different biochemical properties (14). The adenosine triphos- phatase of rabbit white muscle myosin has a higher specific activ- ity (1, 2), a greater alkaline stability (3), and is more sensitive to trypsin (4) than the ATPase from red muscle myosin. In other studies not involving the catalytic site, it has also been shown that acetylation of rabbit red muscle myosin releases small pro- tein units which differ electrophoretically from those released from white myosin (5).

Ina,smuch as the rate of ATP hydrolysis by myosin is probably a limiting step in the speed of muscle contraction (6, 7), differ- ences between myosin ATPases from fast and slow muscles may be of basic importance. In the present studies on cat muscle myosin, special attention is focussed on some new observations relating to (a) the acid lability of fast muscle ATPase, (b) the low degree of activation of ATPase by p-chloromercuribenzoate, and (c) the irreversibility of p-chloromercuriphenylsulfonate inhibi- tion of fast muscle myosin ATPase. In addition, it is shown that CMSI releases two closely associated proteins from fast muscle myosin, and that these closely associated proteins have a definite relationship to myosin ATPase activity.

1 The abbreviation used is: CMS, p-chloromercuriphenylsul- fonate.

METHODS

Myosin Preparation-The flexor hallucis longus and biceps femoris (fast or white) muscles and the soleus (slow or red) muscle of adult cats and rabbits were minced and homogenized in 10 vol- umes of 0.15 M KCl-0.01 M histidine-0.005 M EDTA (pH 6.8) for 90 set in a Sorvall homogenizer. The homogenate was centri- fuged at 1,000 x g for 15 min to separate the myofibrils, which were resuspended and washed three times in 0.15 M KCI-0.01 M

histidine (pH 6.8) by repeated centrifugation at 1,000 x g. The washed pellet was resuspended in 1.0 M KCI, pH 7.0, and stirred for 2 hours at 4’ to extract actomyosin (8). The supsension was then centrifuged for 20 min at 1,000 X g, and the supernatant was made 5 mu with respect to MgCls and ATP in order to dissociate actin from myosin. Actin was removed by centrifugation for 2 hours at 100,000 x g (9)) and any remaining actin and actomyo- sin was removed by precipitation with ammonium sulfate at 35% saturation. The supernatant was dialyzed 15 to 20 hours against 0.05 M KCl-0.005 Y histidine, pH 7.0, until all ammonium sulfate was removed as indicated by the lack of precipitate formation when barium hydroxide is added to the dialysate. The precipi- tated myosin was resuspended in 0.5 M KCl. These preparative procedures were performed at 3”. Further reprecipitation of the myosin was not routinely carried out as this did not alter any of the results. Myosin was also prepared by a modification of the Szent-Gyorgyi method as described by Perry (10). When indi- cated, chromatography of myosin was done by the method of Harris and Suelter (11). Protein was measured by a biuret method.

Preparation of Closely Associated ProteinsMyosin closely as- sociated proteins were dissociated by the addition of 8 moles of CMS per lo6 g of myosin in 0.5 M KU-O.005 M Tris, pH 7.5, at 26”. After 1 hour, or as designated, the remaining myosin was precipitated by dilution of the solution to 0.05 M KC1 either with water or by dialysis of the solution against 0.05 M KU, pH 7.1. The precipitated myosin was separated by centrifugation as the closely associated proteins remained soluble at 0.05 M KCl. Some early experiments done with p-chloromercuribenzoate gave iden- tical results as those with CMS. In addition, alkali closely as- sociated proteins of myosin were prepared by exposure of myosin to pH 10.5 (by addition of 0.1 M glycine-KOH buffer) for 10 min followed by dialysis against 0.03 M KCl, pH 7.0, with separation of myosin and smaller protein units as described above.

Polyacrylamide gel electrophoresis was carried out as described

219

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220 Slow and Fast Muscle Myosin Vol. 245, No. 2

TABLE I Properties of slow and fast muscle myosin ATPase

Details of ATPase assay are given under “Methods.” In Part E, myosin is exposed to pH 10.5 (by addition of 0.3 m&r glycine) for 10 min before being assayed at normal conditions. In Part F, myosin pH is adjusted to 4.35 with 0.3 mM acetic acid on pH-stat at 26” for 5 min before being assayed at normal conditions.

A. Ca++-ATPase (APi, pmoles per mg of protein per min).

B. KCl-EDTA ATPase (APi, pmoles per mg of protein per min)

C. Activation by CMS (%) D. Moles of CMS per lo5 g of myosin

causing maximal ATPase activa- tion.............................

E. Normal Ca++-ATPase activity lost after 10 min at pH 10.5 (%). .

F. Normal Ca*-ATPase activity lost after 5 min at pH 4.35 (70). .

a Standard error of the mean. b Coefficient of variation.

--

-

- I Slow muscle myosin

No. of animals

19

6 8

-

_-

-

Mean

0.22 f 0.016"

1.04 rt 0.07" 320 f 163b

3.4 * 0.275a

63.3 f 9.31b

47.6 f 4.93b

by Ornstein (12) and Davis (13). The stacking gel (10 x 5 mm) of 5% polyacrylamide and the separating gel (60 x 5 mm) of 7.570 polyacrylamide were made up in 8 M urea. The upper stacking gel was adjusted to pH 6.7, the lower separating gel was adjusted to pH 9.6. Bromphenol blue was used as a tracking dye. After electrophoresis, the gels were fixed in 12% trichlor- acet,ic acid for 30 min, stained in 0.077 Coomassie blue dye for 1 hour, and destained for 24 hours in several changes of 7 y0 acetic acid.

Solutions ati Reagents-411 solutions were prepared with dis- tilled, deionized water. Urea was obtained from Baker and Adamson, ammonium sulfate from Mann, p-chloromercuribenzo- ate from Calbiochem, p-chloromercuriphenylsulfonate from Sigma, and electrophoretic materials from Canalco.

Enzymatic Assay-ATPase measurements were carried out at pH 7.8 at 26” in a Radiometer pH-stat in a total volume of 2.0 ml containing either 0.05 M KCl-0.01 M CaC&-0.005 M K&TP or 0.5 M KCl-0.5 mM EDTA. The base used for titration was 0.05 M KOH, and the other reagents added to the ATPase assay are de- scribed later.

RESULTS

Myosin A TPase

Table I, A to F, summarizes the observations made on cat myo- sin ATPase. The fast muscle myosin Ca*-stimulated ATPase activity is about four times that of slow muscle myosin (Part A) and its KCl-EDTA-ATPase activity is about twice that of slow muscle (Part B). In the presence of optimal CMS concentra- tions, slow muscle myosin Ca*-ATPa.se was increased 3210/, wherea,s fast muscle myosin ATPase was increased only 44yo (Part C) ; in general, it took more CMS to activate slow muscle myosin ATPase than to activate fast muscle myosin ATPase maximally (Part D) .

Exposure of slow and fast muscle myosin to pH 10.5 for 10 min at O-3” was without effect on Ca*-ATPase activity, but if the myosin was exposed to alkaline pH at room temperature for this time slow muscle myosin lost 64% of its normal Ca*-ATPase

Fast muscle myosin

16

5 B

-

L

1Mean

0.80 + 0.081"

1.99 + 0.253= 44 f 25*

2.4 r!z 0.245"

9.86 xt 6.42b

83.6 i 4.83b -

p (from 1 test), slow VWSUS fast

<O.OOl

<O.OOl

<0.025

a,ctivity while fast muscle myosin lost only 6% of its normal ac- tivity (Part E). Exposure of slow and fast myosin to pH 4.35 (regulated in pH-stat with 0.25 to 0.5 mM acetic acid) for 5 min caused slowmyosin to lose47.6% of its ATPase activity; fast myo- sin lost 83.6% of its ATPase activity (Part F). Similar qualita- tive observations have been made on the acid and alkali lability of actomyosin ATPase of cat fast and slow muscle (14).

Proteins Released by CM&-The treatment of myosin in 0.5 M KC1 with 8 moles of CMS per lo5 g of myosin releases proteins which are soluble in 0.05 M KCl. Fig. 1 shows that disc elec- trophoresis in 8 M urea on polyacrylamide gel resolves the closely associated proteins released from fast muscle into four bands, A, B, C, and D. Electrophoresis of the closely associated pro- teins released from slow muscle myosin resolves only two bands, B and D. Gel 3 in Fig. 1 shows that Bands B and D from slow and fast muscle myosin are electrophoretically identical. Chro- matography of myosin successively on cellulose phosphate and DEAE-cellulose results in the removal of Bands B and D so that CMS releases only Bands A and C from fast muscle myosin and no electrophoretically identifiable proteins from slow muscle myosin (Fig. 2). Because Bands B and D in our studies resem- bled tropomyosin and troponin bands on similar studies by others, these proteins were isolated from native actomyosin ac- cording to the method of Schaub, Hartshorne, and Perry (15). Electrophoresis of these proteins in our system resolved two bands which had the same Rp values as our Bands B and D.

Comparative studies were done on fast muscle myosin pre- pared according to the Szent-Gyorgyi method. Electrophoresis of closely associated proteins released by CMS resulted in the resolution of two bands with identical Rp values to Bands A and C (Fig. 2).

Relation between Closely Associated Protein Release, CMS Titration, and

ATPase Activity

Incubation of fast muscle myosin in the presence of 3 to 6 moles of CMS per lo5 g of myosin releases large amounts of A

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Issue of January 25, 1970 F. J. Samaha, L. Guth, and R. W. Albers 221

FIG. 1 (upper). Disc electrophoresis of myosin CMS closely associated proteins on polyacrylamide gel in 8.0 M urea. 1, cat white muscle; I, cat red muscle; 3, mixture of cat red and white muscle; 4, rabbit white muscle; 5, rabbit red muscle; 6, mixture of rabbit white and red muscle; 7, mixture of cat and rabbit white muscle; 8, mixt,ure of cat and rabbit red muscle. Myosin in 0.5 M KC1 was treated with 8.0 moles of CMS per 105 g of myosin for 60 min (26’). The myosin was precipitated by dialysis against 0.03 M KCl. An aliquot of supernatant corresponding to about 1 mg of myosin was used for electrophoresis.

FIG. 2 (lower). Disc electrophoresis of myosin CMS closely as- sociated proteins on polyacrylamide gel in 8.0 M urea. Top, chromat,ographed myosin from cat white muscle; middle, chro-

CMBS (moles/lO~ myosin)

FIG. 3. Effect of p-chloromercuriphenylsulfonate (Cn/rSS) on ATPase activity and release of closely associated proteins from fast muscle myosin. O-O, percentage of normal ATPase activity. Myosin in 0.5 M KC1 was treated with CMS for 3 min at 23” followed by estimation of ATPase activity in 0.05 hc KCl- 0.01 M CaClz-0.005 M KzATP, pH 7.8, at 26”. O-O, percentage of CMS closely associated proteins released. Myosin in 0.5 M KC1 was treated with CMS for 60 min followed by precipitation of residual myosin and estimation of closely associated proteins in the supernatant.

and C, whereas 0 to 2 moles of CMS per lo6 g of myosin releases none to little closely associated proteins (Fig. 3). Treatment of the soleus muscle myosin with CMS releases no closely associ. ated proteins and does not alter ATPase activity even if the myosin is thrice treated with CMS (Table II). In the case of fast muscle myosin, however this treatment removes Bands A and C and results in a two thirds loss of ATPase activity of the remaining myosin (Table II).

Fig. 3 also shows that in white muscle myosin, little or no closely associated proteins are released in the presence of the relatively low concentrations of CMS that cause activation of ATPase activity; moreover the amount of closely associated proteins released is inversely related to the degree of loss of ATPase activity. The total amount of closely associated pro- teins released from myosin prepared either by extraction from myofibrils or by the Szent-Gyorgyi method is about 5 to 10% of the total myosin protein. Fig. 4 further documents the rela- tionship between the loss of ATPase activity and the release of closely associated proteins. The loss of ATPase activity and the release of closely associated proteins in the presence of CMS follow a similar but inverse temporal pattern.

Cat Myosin Alkali Closely Associated Proteins

Since much work has been done on the closely associated pro- teins released from rabbit muscle myosin treated with weak alkali (16-22), observations on cat myosin alkali closely associ- ated proteins are presented. As found by others, the myosin,

matographed myosin from cat red muscle; bottom, cat white muscle myosin prepared by a modification of the Szent-Gyorgyi method. Myosin in 0.5 M KC1 was treated with 8.0 moles of CMS per 106 g of myosin for 60 min (26’). The myosin was precipitated by dialysis against 0.03 M KCl. An aliquot of supernatant cor- responding to about 1 mg of myosin was used for electrophoresis.

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222 Slow and Fast Muscle Myosin Vol. 245, No. 2

after removal of alkali closely associated proteins, showed no ATPase activity. Electrophoresis of the alkali closely associ- ated proteins in 8 M urea followed by staining with a relatively insensitive dye such as amido black resulted in the appearance of 4 to 5 bands. But if the more sensitive Coomassie blue dye

TABLE II E$ect of CM&protein removal on myosin ATPase

The CMS protein is dissociated from myosin upon the addition of 8 moles of CMS per 106 g of myosin with subsequent isolation as described under “Methods.” Mercaptoethanol was added in 1000 times excess over original amount of CMS added. See “Methods” for details of ATPase assay. These results are repre- sentative of a series of experiments performed under various conditions.

Ca++-ATPase activity, APpi

I. Fast muscle myosin Normal......................... CMS closely associated proteins removed. . . . .

II. Slow muscle myosin Normal......................... CMS closely associated proteins removed. . . .

1.40

0.48

0.26

0.26

t

I., 30

20

i

J

J 20 40 60 80 100 120 140 160 18 hrs

MINUTES

FIG. 4. Effect on calcium ATPase activity and release of closely associated proteins by 8 moles of CMS per lo6 g of myosin with reference to time. O-O, percentage of normal slow muscle myosin ATPase activity; O----O, ‘percentage of normal fast muscle myosin ATPase activity. Myosin in 0.5 M KC1 was pre- viously incubated in CMS for stipulated times before assay. See “Methods” for details of assay. A--A, percentage of CMS closely associated proteins released. Myosin in 0.5 M KC1 was treated with CMS for stipulated times before removal of residual myosin and estimation of closely associated proteins in the supernatant. See “Methods” for details.

FIG. 5. Disc electrophoresis of myosin alkali closely associated proteins on polyacrylamide gel in 8.0 M urea. 1, cat white muscle; 2, cat red muscle; 3, rabbit white muscle; 4, rabbit red muscle. Myosin in 0.5 RI KC1 was incubated at pH of 10.5 for 10 min (23”). The myosin was precipitated by dialysis against 0.03 M KCl. An aliquot of supernatant corresponding to about 1 mg of myosin was used for electrophoresis.

was used, 8 to 10 bands appeared and the very high background staining suggested the presence of still more proteins not yet re- solved (Fig. 5). Electrophoretic differences were noted between fast and slow muscle myosin alkali closely associated proteins. Although some bands from alkali and CMS closely associated proteins seemed identical, the presence of many bands in the former prevented us from proving this point conclusively.

The question as to whether the many alkali closely associated proteins were derived from degradation of the CMS closely associated proteins led to the following experiment. The CMS closely associated proteins of fast muscle myosin were exposed to a pH 10.5 medium for 30 min followed by a return of the pH to 7.0. Subsequent electrophoresis showed no change in elec- trophoretic pattern, and it is therefore concluded that the mul- tiple bands of the alkali closely associated proteins are not de- rived from the closely associated proteins released by CMS.

Comparison of Cat and Rabbit CMX and Alkali Closely Associated Proteins

Fig. 1 shows that upon electrophoresis, the CMS closely as- sociated proteins from both slow and fast muscle myosin of the rabbit resolve into four bands. Electrophoresis of a mixture of rabbit slow and fast myosin subunits shows Band y to be con- fined to slow muscle myosin closely associated proteins and Band z to those of fast muscle myosin. The three remaining bands, B, C, and D, are electrophoretically identical. Since Bands B and D from the cat myosins are removed by chroma- tography, it is likely that the distinctive CMS proteins of the rabbit myosin are Bands x and C from fast muscle and Band, y from slow muscle. The small amount of C in rabbit soleus cor- relates with the more heterogeneous fiber population relative to cat soleus (14).

Electrophoresis of a mixture of CMS closely associated pro- teins from rabbit and cat soleus muscle myosin indicates that Bands y and C from the rabbit are confined to this species (Fig.

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Issue of January 25, 1970 F. J. Xamaha, L. Guth, and R. W. Albers 223

1). Electrophoresis of a mixture of rabbit and cat fast muscle myosin CMS closely associated proteins shows that the slowest moving bands, x and A, respectively, are different in the two species, while Band C is electrophoretically identical (Fig. 1).

As in the case of cat myosin, electrophoresis of rabbit alkali subunits in 8 M urea followed by staining with Coomassie blue results in the appearance of so many bands that comparisons between the two species are not readily made.

DISCUSSION

The following properties of fast and slow muscle myosin ATPase of the cat have been documented in the present work. The fast muscle myosin ATPase of the cat exhibits a higher ac- tivity, a greater alkaline stability, and a maximal activation by smaller amounts of CMS. In addition, slow muscle myosin ATPase of the cat is more susceptible to CMS activation and is more acid-stable than is fast muscle myosin ATPase of the cat. More details of the acid stability of slow muscle myosin ATPase of the cat are presented elsewhere (14). Some of these observa- tions have been presented in the case of rabbit muscle (l-3). It should be appreciated that muscles are composed of fibers of various types, and the above alkali-acid stability studies pre- sumably show an algebraic summation of the various types of fibers in each myosin preparation. Histochemical studies re- veal that single muscle fibers of diierent types show an even more striking acid-alkali differential lability of their actomyosin ATPase (14).

It is well known from studies on rabbit myosin that ATPase activity is inhibited by high concentrations of CMS (23-26). The present work shows that in the case of cat slow muscle myosin this inhibition is complet,ely reversed by removal of the CMS, but it is not reversible for fast muscle myosin ATPase. This observation plus the known temperature dependence of the CMS effect on myosin (26) may explain why some investi- gators showed complete recovery (23, 24) of ATPase activity after CMS inhibition, while others (25, 26) observed very little recovery of Ca*-ATPase activity with mercaptoethanol treat- ment.

The first observation that a mercurial agent might release pro- tein from myosin was made by Kominz, who used the analytical ultracentrifuge in his studies (27). He noted that when sufE- cient methylmercuric hydroxide was added to inhibit myosin ATPase activity, low molecular weight protein was released. Locker and Hagyard (5) showed that acetylation of slow and fast muscle myosin from the rabbit releases different proteins as determined by polyacrylamide gel electrophoresis. Although acetylation results in loss of ATPase a&iv&y of slow and fast myosin (5), in the present experiments, CMS released closely associated proteins from only fast muscle myosin, and it is only this myosin which loses some ATPase activity. Since the rate of loss of ATPase activity inversely paralleled the rate of release of closely associated proteins in the presence of CMS and since the amount of closely associated proteins released inversely paralleled the degree of inhibition of ATPase activity by various concentrations of CMS, we suggest that the presence of closely associated proteins are probably necessary for maximal ATPase activity of fast muscle myosin. Further work is presently being done to establish more clearly the relationship of ATPase ac- tivity to the closely associated proteins. We found, as did Gaetjens et aZ. (21), that addition of closely associated proteins

back to either CMS-treated myosin or to normal myosin effects no changes in the myosin ATPase activity.

Tonomura and Furuya (28) and Kitagawa, Yoshimura, and Tonomura (29) showed that p-chloromercuribenzoate causes release of calcium and magnesium from myosin. Moreover, Tonomura, Yoshimura, and Kitagawa (30) showed that acto- myosin prepared from actin plus divalent cation-free myosin has different biochemical properties from actomyosin prepared from actin plus normal myosin. Many different proteins and enzymes can be fragmented by p-chloromercuribenzoate (31-41). One of the more interesting studies has been the work on aspar- tate transcarbamylase (39) in which p-chloromercuribenzoate treatment splits this enzyme into separate catalytically active and regulatory proteins. These data plus the data showing the CMS closely associated proteins are released only from fast muscle myosin, with an associated loss of ATPase activity, raise the question as to whether they represent regulatory proteins for the function of actomyosin.

Ac~nowledgmentsCompetent technical assistance was pro- vided by Mr. William Brown and Mrs. Phyllis Watson.

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Frederick J. Samaha, Lloyd Guth and R. Wayne AlbersPROTEINS BY p-CHLOROMERCURIPHENYLSULFONATE

TRIPHOSPHATASE ACTIVITY AND RELEASE OF ASSOCIATED Differences between Slow and Fast Muscle Myosin: ADENOSINE

1970, 245:219-224.J. Biol. Chem. 

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