Vol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 245, No. 1, Issue of January 10, PP. 15-22, Printed in U.S.A.

1970

Studies

on the Subunit
JOSEPH GAZITH,~ SYLVIA

Structure
AND

of Myosin*
(Received for publication, October 31, 1968) WILLIAM

HIMMELFARB,

F. HARRINGTON

From the Department of Biology, McCollum-Pratt Institute, the Johns Hopkins University, Baltimore, Maryland 21218

SUMMARY The low molecular weight protein associated with rabbit skeletal myosin has been estimated by gel titration studies in 5 M guanidine hydrochloride and was found to constitute 9 f 2% of the myosin mass. We have found that 40 to 50% of the low molecular weight protein can be removed from myosin without any apparent changesin the adenosinetriphosphatase activity. Attempts to remove a larger fraction of low molecular weight protein resulted in a concomitant loss of enzymatic activity. The molecular weight of the major myosin subunit freed of low molecular weight protein has been redetermined in 5 M guanidine hydrochloride and was found to be 194,000 g per mole. The significance of its presence in assessing the molecular weight of the major subunit at low speed and high speed sedimentation equilibrium has been investigated.

ment 1 yielded a recovery of 42y0. Moreover, the actin-binding capacity of these reconstituted systemsseemed to follow the ATPase activity. In this work, we have attempted to dissociate low molecular weight protein selectively from myosin under conditions which would retain the enzymatic activity of the native protein. As we will show, only a fraction of the LMP mixture (about 50y0) is required for full ATPase activity. We have also determined the molecular weight of LMP-free myosin in 5 M guanidme hydrochloride because previous determinations(3, 7, 11, 13, 14) of the size of the major subunit in this solvent have been carried out on the myosin-LMP mixture.2
MATERIALS AND METHODS

The presence of low molecular weight protein, or light chains, 1in myosin preparationshasbeenclearly established by a variety of techniquesin recent years (l-5). LMPl isolated by various methodshas been shownto be heterogeneous by polyacrylamide and celluloseacetate gel electrophoresis(6-8) and column chromatography (9), and the weight fraction of LMP associated with myosin has beenreported to be between5% (9) and 15% (2), with molecular weights ranging between 20,000 (7, 8) and 32,000(10) on the unfractionated LMP mixture. On the basisof the finding that LMP has an unusually strong affinity for myosin, and that it occursin all myosin preparations,it has beenpostulatedas a subunit of the molecule(3, 5, 7, 11). This proposalhas beengiven strong support by the recent studiesof Stracher (12), who hasshownthat rapid separationof low molecular weight protein from the heavy chainsof myosinon Sephadex G-200 columnsequilibrated with 4 M LiCl, followed by remixing and dialysis,resultsin about 30% recovery of the original ATPase activity of native myosin. Similar experimentswith Subfrag* This work is Communication 574 from the Department of Biology, the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland, and was supported by United States Public Health Service Grant AM-04349. $ Present address, Department of Microbiology, Bar Ilan University, Ramat Gan, Israel. 1 The abbreviations used are: LMP, low molecular weight protein; DTNB, 5,5-dithiobis(2-nitrobenzoic acid).

Myosin was preparedby the method of Kielley and Bradley (16) unlessotherwise noted and included, as a final step, ammonium sulfate fractionation between40% and 50% saturation. For resolution of LMP and myosin by gel filtration, Sephadex G-100 gel wasequilibrated with 5 M guanidinehydrochloride and poured into a column (2.5 x 100 cm) which was adapted for reverseflow to prevent packing of the gel. Myosin wasallowed to react with a lo-fold molar excess (with respectto -SH groups) of N-ethylmaleimide overnight at 5 (13), and the resulting solution, made 5 M with respectto guanidinehydrochloride, was applied to the column at a concentration of lessthan 0.5%. Higher protein concentrationsresulted in poor resolution of the elution patterns because of the high viscosity of the sample. For reversible blocking of -SH groups, myosin was allowed to react with 0.01 M DTNB or p-chloromercuribenzoate at pH 8.5 in 0.5 M KCI-0.01 M Tris-HCI. When DTNB was used,0.01 M EDTA wasalways present. In somecases, the reaction was carried out in the presence of 1 M or 2 M urea. Removal of the dissociated LMP wasachieved by dilution-precipitation of the myosin at an ionic strength of 0.05. The blocking reagentwasremovedand the sulfhydryl groupswere regenerated by dialysis against 0.5 M KCl, 0.01 M EDTA, and 0.01 M /3mercaptoethanolor dithiothreitol, pH 7.0 to 8.5. The extent of sulfhydryl regenerationwas assayed with DTNB in 1 M guanidine hydrochloride-O.01M Tris-HCl-0.01 M EDTA, pH 8.5
2 Since this paper was submitted, two reports have appeared in which the molecular weight of the major subunit was determined with the use of LMP-free myosin in 5 M guanidine hydrochloride. A value of 232,000 to 238,000 was estimated by Frederickson and Holtzer (10) from viscosity measurements alone, and Gershman et al. (15) obtained 212,000 using high speed sedimentation equilibrium.

.5

16

Subunit Xtructure

of Myosin

Vol. 245, No. 1

(17). Guanidine hydrochloride was used to accelerate the time required for complete reaction. Irreversible blocking of -SH groups was accomplished by causing myosin to react with Nethylmaleimide as described previously. All chemicals were reagent grade except as noted. Guanidine hydrochloride was prepared from guanidine carbonate by the method of Anson (18) and dried in a vacuum desiccator over Prior to use, the guanidine hydrochloride was examined P&s. for the presence of ultraviolet-absorbing materials by comparing a solution of 7 M guanidine hydrochloride against water. Urea was recrystallized from 70% ethanol solutions. Calcium-activated ATPase activity of myosin was determined as before (16); the activity in the presence of EDTA was estimated according to the method of Kielley, Kalckar, and Bradley (1%. LMP-free myosin was prepared for molecular weight studies by treating the native protein overnight at 5 with N-ethylmaleimide, followed by gel filtration on a Sephadex G-150 column (4 x 120 cm) which had been previously equilibrated in 3 M urea-O.5 M KCI. The eluted LMP-free protein was adjusted to 5 M with respect to guanidine hydrochloride and dialyzed against 5 M guanidine hydrochloride for 24 to 48 hours. The resulting myosin solution was centrifuged at 50,000 rpm for 2 hours, and the protein concentration was adjusted to that required for equilibrium sedimentation. This protein solution was redialyzed against 5 M guanidine hydrochloride for an additional 72 to 96 hours and transferred directly to the centrifuge cell. Protein concentrations of native myosin in 0.5 M KC1 were estimated from an extinction coefficient of 500 cm2 per g (at X = 280 mp) determined previously (20). The extinction coefficient of LMP-free myosin in 5 M guanidine hydrochloride (535 cm2 per g at X = 280 rnpcl)was determined by the method of Kielley and Harrington (13). Solutions were prepared by weighing the lyophilized protein into 5 M guanidine hydrochloride. The amount of water bound to the protein was obtained by drying triplicate samples under vacuum over PzOs at 110. The apparent partial specific volume, $ (defined according to Casassa and Eisenberg (21)), of LMP-free myosin in 5 M guanidine hydrochloride was determined after 4 days of dialysis at 20. Densities of protein solution and dialysate were measured with 25-ml pycnometers, and the protein concentration was determined from measurement of the optical density (h = 280 rnp) with the use of the extinction coefficient of LMP-free myosin in 5 M guanidine hydrochloride. The extinction coefficient of LMP in 0.5 M KC1 was determined on material isolated from Sephadex G-150 columns equilibrated with 5 M guanidine hydrochloride. Solutions were dialyzed exhaustively against water, and the protein concentration was estimated from Kjeldahl analysis with the use of a nitrogen-protein factor obtained from ammo acid analysis. The extinction coefficient of LMP in 5 M guanidine hydrochloride was obtained by comparing the optical density of a solution prepared by dilution of an aliquot of LMP plus solid guanidine hydrochloride with 0.5 M KC1 to an equivalent aliquot made up to the same volume with 0.5 M KC1 alone. Extinction coefficients for LMP in 0.5 M KC1 and 5 M guanidine hydrochloride were identical (500 cm2 per g at X = 276 mp).a
efficient for LMP (360cm2per g) than is reported in this paper (500cm2 per g). If the lower extinction coefficient is used, the amount of LMP bound to myosin is raised to 12.5 f 2%.

Molecular weights of LMP-free myosin in 5 M guanidinehydrochloride were estimated by both the high speedmeniscus depletion technique of Yphantis (22) (22,000rpm) and the low speedsedimentationequilibrium method (9,000to 10,000rpm). Temperature was always 20. For selectionof the appropriate speed,the parameter a,,, = (M(1 - @p)w3/2 RT was used, wherep is the density of the solution and w is the angular velocity. On the assumption that M is 200,000g per mole,the speed wasselected so that a, = 4.5 to 6.5 cm+ for high speed equilibrium and 1.5 to 2.5 cmm2 for the low speedruns. Equilibrium wasestablished after 24 to 48 hours,dependingon the speed and column height, and wascheckedfor both methodsby comparing the gradientson two plates taken at least 24 hoursapart. Column heights were 2 to 2.5 mm. Molecular weights were calculated as a function of concentration in the cell, by a computer program written by Roark and Yphantis (23). After equilibrium was establishedand recorded in the low speedexperiments,the speedwas doubled and the decrease in concentration near the meniscus was followed with time according to a modification of the procedureof LaBar (24) (seeGodfrey (25)), until completedepletion at this level of the cell occurred. To ensurecomplete depletion at the meniscus,the speedwas elevated to 35,000 rpm at the end of the experiment, and the concentration near the meniscus wasfollowed further with time. In this way, the concentration near the meniscus could be determined to an accuracy of about 0.03fringe and the concentration at each point in the cell could be established. Although this method requires reading many photographic plates, it is independent of the assumptionof conservation of mass. The same procedurewas adopted in the high speedexperimentsto ensure completedepletion of the meniscus. Weight averagemolecular weights were calculated for each point in the cell by the equation (1) where c is concentration and r is the distancefrom the center of rotation. The z-average molecularweight at each point in the cell is calculated in the Roark-Yphantis computer program by the relation

(2)

and the number averagemolecularweight by the relation M,(r) = 49 de c(m) -+sm Kh9 M,(m) (3)

3$!%racher (12) has estimated a somewhat lower extinction co-

where c(m) and M,(m) are the concentration and number averagemolecularweight at the meniscus. The term (c(m))/(lll,(m)) in Equation 3 can be neglectedin high speedexperimentswhen c(m) is essentiallyzero. In low speedexperiments,when c(m) is not equal to zero, the concentration at the meniscus is determined as mentionedabove and the problem is to choosea value for M,(m). This can be approximated by a linear combination of M,(m) and M,(m), so that (23)

Issue of January

10, 1970

J. Gazith,

X. Himmelfarb,

and W. F. Harrington

17

Amount of
MYOSIN

small

TABLE I protein bound to myosin estimated by two methods Small protein

Sample

and method

of myosin

preparationa

h
0 0.0 --.-.-.-..--. I 0 20 II
40 60

j II/I
80 100 120 140

Ld--. III
160 180

. .._ ~ _.____ I
200 220

j
240

EFFLUENT

VOLUME

(ml)

FIG. 1. Gel filtration of myosin on a Sephadex G-100 column in 5 M guanidine hydrochloride. Applied, 8 ml of 0.5% myosin in 5 M guanidine hydrochloride. The column was developed with a flow rate of 8 ml per hour. Fractions of 5 ml each were collected. Inset, to 43 mg of myosin in 5 M guanidine hydrochloride, 5 mg of ribonuclease were added (Wort.hington, ethanol-fractionated). The mixed protein solution was applied to the column and eluted as above.

1. Regular. ............................... 2. Regular. ............................... 3. Regular. ............................... 4. Regular. ............................... 5. Regular. ............................... 6. Regular. ............................... 7. Regular ................................ 7. Regular, no (NH4)2504 .................. 8. Regular, pH 6.5 ......................... 9. Regular, 5 min .......................... 9. Regular, 15 min ......................... 9. Regular, 40 min ......................... 10. Potassium phosphate, pH 9.3, 0.50 M. .... 11. Potassium pyrophosphate, pH 9.3,0.26 M. Average ..................................
I

9.5

5.9

7.7 9.0 7.0 10.1 10.0 9.7 8.0 7.4 10.0 7.4 10.3 9.2 9.9 8.9

7.1 7.2 6.3 5.7 6.4

6.4

a A regular preparation is extraction of muscle mince in 0.5 M KCl-0.1 M K2HP04, pH 9.3, extracted for 10 min unless otherwise noted. measurement. The amount of LMP associated with myosin estimated in this way was invariably lower than that determined by gel filtration (Table I), presumably as a result of its coprecipitation with myosin. When the precipitated protein was redissolved in pH 11.5 buffer (2) and examined at high speed in the ultracentrifuge (see below), approximately 2 to 3% of the total protein was always present as LMP. Thus, these experiments, when taken in conjunction with the gel filtration studies, indicate that about 8 to 10% of LMP is strongly associated with the native myosin molecule as judged by this criterion. Effect of Varying Extraction Conditions on Amount of Low Molecular Weight Protein Associated with Myosin-The effect of varying the composition of the extraction medium on the amount of low molecular weight protein was also investigated in a series of high speed ultracentrifugation experiments of myosin at pH 11.5 in a solvent consisting of 0.4 M KCI-0.1 M Na&Oa. Under these conditions, as was originally shown by Kominz et al. (2), LMP is released and is clearly resolved in the schlieren pattern from the more rapidly sedimenting myosin peak. Measurements of the area of the LMP peak showed it to be directly proportional to the loading myosin concentration and, consequently, no correction was made for a Johnston-Ogston effect in estimating the concentration of this component. (Inasmuch as only relative, not absolute concentrations are measured by this procedure, any error resulting from the neglect of the Johnston-Ogston correction should be negligible.) The area of the LMP peak at pH 11.5 was compared to that found in a standard (Kielley-Bradley) myosin preparation (assumed as 100% LMP) which was centrifuged at the same time in a companion 12-mm double-sector ultracentrifuge cell. The compositions of the various extraction media are given in Table II. Myosin was also extracted from myofibrils prepared from the back muscle of rabbit and stored in 50% glycerol at -20 for over 1 year. These were washed exhaustively to remove soluble proteins and were used to extract myosin by the Kielley-Bradley method. In all of these prep-

M&n) = t M&n) - + K(m)

(4)

The number average molecular weight so obtained is an approximation because of the assumptions involved in Equation 4; however, the error in M,(m) does not seem serious, because values obtained at high speed (c(m) = 0) and low speed (c(m) = 0.2 to 1.2 mg per ml) are closely similar.
RESULTS

Amount of Low Molecular Weight Protein Associated with Myosin-Although low molecular weight material is not easily detected in velocity sedimentation studies of myosin in the dissociating solvent 5 M guanidine hydrochloride, gel filtration elution patterns in this solvent system clearly show the presence of a minor component (LMP) trailing behind the major subnuit peak. As can be seen from inspection of Fig. 1, this component elutes ahead of ribonuclease, which suggests that the molecular weight is probably greater than 14,000 g per mole. For estimation of the relative amount of bound low molecular weight protein released when the polypeptide chains of myosin are unfolded and dissociated in guanidine hydrochloride, the area of each peak of the bimodal elution pattern was measured and the concentration of each component was estimated from its extinction coefficient (see Materials and Methods). The average amount of low molecular weight protein dissociated from myosin under these conditions approximated 9% of the total protein mass and seemed to be independent of the time of extraction or of the solvent conditions used in the isolation of myosin (Table I)? For a quick, routine assay of the amount of bound low molecular weight protein, myosin was incubated in 2 M urea (0.5 MKCI-0.01 M EDTA) overnight at room temperature. This treatment also results in dissociation of LMP from myosin. Dilution of the resulting solution with 2 M urea to an ionic strength of 0.05 precipitated the myosin, leaving LMP in the supernatant where its concentration could be determined by optical density

18
TABLE

Subunit Structure of Myosin

II

Vol. 245, No. 1

Compositions of various solvents used for extracting myosin

Af

KC1
KpHP04 KC1 KzHPOd KzHPO4 KC1 &CO, KHCOs KzHPOd

1
0.1 2 0.1 0.226 0.6 0.01 0.04
0.226 0.226

9.3 9.3 9.3 9.3 10.8

6.5

KaHP04

arations,the amount of LMP released from myosinwasconstant and independentof the extraction mediumcomposition. The apparent invariance in the amount of low molecular weight protein associated with myosin isolated in the presence of a wide variety of extracting solvents suggests, in agreement with the conclusions of earlier workers, that this material may be an essential componentof the myosin structure.

Effect of Removal of Low Molecular Weight Protein on ATPase

Activity----The methodsusedso far for the removal of LMP also result in denaturation of the myosin molecule,and a searchwas initiated for relatively mild conditionsthat would dissociate low molecularweight material in the absence of significantalterations in ATPase activity. Because the ATPase activity of myosin is known to be intimately dependent on specific -SH groupswithin the globular region of the molecule(26-28), a generalprocedure wasdevelopedbasedon reaction of all of the thiol groupsof the protein with a removable blocking reagent. LMP is then released from myosin in the presence of a dissociating solvent (see below), and the residual myosin is precipitated at low ionic strength. The blocking groups are then removed by dialysis against @-mercaptoethanol or dithiothreitol and the resulting myosin examinedfor ATPase activity and residualLMP. Reaction of the sulfhydryl groupsof myosin with the removable blocking reagentsp-chloromercuribeneoate or DTNB alone results in a bimodal schlierenpattern in the ultracentrifuge at high speed. The slowcomponentshowed sedimentation characteristics similar to thoseof the LMP peak observedat pH 11.5. No lossin ATPase activity was observedafter precipitation of the myosinfollowed by regeneration of the -SH groups. Centrifugation in alkali indicated the removal of 15 to 20% LMP. For further enhancement of the dissociation of low molecular weight protein, myosin was allowedto react with DTNB in the presence of varying concentrationsof urea. Urea was used as the dissociating solvent of choicebecause it has beenpreviously shown to be a rather mild denaturing agent for myosin at low concentrations(29). We found incubation of myosinfor 15 min at 4 in 1 M urea-O.01 M DTNB to be the optimum conditionsfor dissociation of LMP and regeneration of ATPaseactivity (Fig. 2). About 40 to 50% of LMP was released under theseconditions, the major fraction of which couldbe removedby precipitation of the myosin with no apparent lossin either Ca++- or EDTA-activated ATPase activity, or in the number of regeneratedthiol groups (Table III). If the sulfhydryl groups are regenerated without removing LMP (i.e. in the absence of the precipitation step), the releasedlow molecularweight material reassociates

FIG. 2 (upper). Ultracentrifugepattern of LMP released afterremyoaction of myosinwith DTNB in the presence of urea. To 1.6a/, sin solution, DTNB wasaddedto a final concentrationof 0.01 M and urea to a final concentration of 1 M. DTNB and urea were similarly added to the buffer. A filled Epon double-sector cell wasusedat 52,000 rpm. Temperature,3; bar angle,70. Convection spikesare the result of the high myosin concentrationin

this experiment. FIG. 3 (lower). Disc gel electrophoresis

of reduced, carboxymeth-

tion removed by treatment with DIWB-2 M urea at 4. Stained with Amido schwarz dye. b, LMP residual fraction removed by treatment of the myosin from a with 2 M urea overnight at room temperature. Stained with Amido schwarz dye. c, same as a, stained with Coomassie brilliant blue (33). d, same ss 6, stained with Coomassie brilliant blue.

ylated LMPfractions in7.5%polyacrylamide,pH9.3. a, LMP frac-

with myosinand subsequent velocity sedimentation studiesshow only a single,hypersharpschlieren peak. Attempts to retain full ATPase activity while dissociatinga larger fraction of LMP in the presence of higher concentrationsof urea have been unsuccessfulin our hands. Attempts to prevent lossof activity by including ATP, Pod-, or Ca++ in the dissociating mediumwere alsounsuccessful. In 1.5 M or 2 M urea (0.01 M DTNB), 70 to 80% of the LMP was removed from myosin, but with a concomitant 70 to 90% lossin ATPase activity. Dissociationof LMP could be reversedby removing the urea and regenerating the sulfhydryl groupsas before, but the depression in ATPase activity was not reversed(Table III). Following reaction with the thiol-blocking reagentsin 2 M urea, only a fraction of these groups could be regenerated,in agreementwith earlier studies

Issue of January

lo,1970

J. Guzith, S. Himmelfarb,

and W. F. Hawington
TABLE

19
IV

TABLE III Effect of removal of small protein on enzymatic activity ojf myosin Preparation protein remaining
SIUall

Amino

activiv

R&tiW

julfhydryl :roups per 106 g of mywin

weight protein isolated by different methods All analyses were on duplicate samples hydrolyzed at 110 for 24 hours. No corrections were made for time-dependent losses.

acid composition

of low molecular

%
Urea, 1
1
M,

I
U2,
0.85

I
C!&romercuribenmate (15-25)

I
DTNB +1x urea (40-45)

I
DTNB +2x urea (70-80) Urea, 2d (residIlaP)

plus DTNB,

0.01

61
59 68 59 54 58

1.00
0.80

2 3 4 Control Urea, 2 M, plus DTNB,!O.Ol 1 3 5 6 Control

100
M

1.00 1.00 1.00 1.00 0.10

6.3 5.9 8.5 6.3 7.3 7.2 6.0 5.3 4.2 4.5 4.5

(col-

DTNB (15-25)

44 46

100

0.31 0.0 0.0 0.15

Q Following removal of DTNB from myosin by dialysis against 0.5 M KCl-0.01 M EDTA and 0.01M ,%mercaptoethanol or dithio-

threitol. Myosin in 0.5 M KC1 was incubated for 15 min in the presence of the dissociatingsystem at 4, then precipitated from
solution by lowering the ionic strength to 0.05 M KC1 either by

adding 2 M urea or water.

(30) in which attempts were madeto regeneratemyosin ATPase 1.0 0.6 activity from the fully dissociatedand unfolded polypeptide chainsin 5 M guanidinehydrochloride. = % LMP removed. Because only about 40 to 50% of low molecularweight protein b Sample reduced and carboxymethylated before analysis (see can be removedfrom myosin without lossof ATPase activity, it Reference 31). 0 Residual is LMP remaining after 2 M urea-DTNB treatment is important to establishwhether the DTNB treatment effectsa and is removed by treatment at room temperature overnight in selectiveremoval of onecomponentor dissociates the wholeLMP mixture to varying degrees. Amino acid analysis of LMP iso- the presence of 2 M urea. lated after reaction with DTNB alone (15 to 25% dissociation), DTNB in the presence of 1 to 2 M urea (4; 40 to 80% dissociaMolecular Weight of Constituent Poplypeptide Chainsof Myosin tion), or LMP isolated by column chromatography in the pres-The weight averagemolecularweight (M,) of the dissociated enceof 5 M guanidinehydrochloride showedno sign&ant trends polypeptide chainsof myosin hasbeen reported in earlier Archi(Table IV), and all showed high phenylalanineto tyrosine ratios bald and sedimentation equilibrium studies (13, 14). These asreported earlier (5, 9). Disc electrophoresis of these preparaexperiments were performed on solutions of unfractionated
tions showed a pattern consisting of three major bands and several minor bands. According to a suggestion of Weeds (31),

Lysine . . . . Histidine . . Arginine . . Aspartic acid. . Threonine. . . .. Serine . . . . Glutamic acid. . Proline. . Glycine. , . . Alanine. Valine . . Methionine . , . Isoleucine . . . Leucine. Tyrosine. . Phenylalanine.. Carboxymethylcysteine. . . . .

8.3 0.7 3.0 13.6 5.3 3.5 16.0 4.0 7.4 9.4 6.3 3.8 4.8 6.3

1.6 6.1

9.3 1.2 3.5 11.4 4.6 4.4 15.3 5.2 7.5 9.5 6.0 3.1 4.7 6.2 1.7 5.2

11.9 0.8 3.9 13.2 5.5 3.6 13.9 3.9 7.8 8.9 5.3 3.3 4.7 5.6 1.3 6.5

10.1 0.9 4.0 12.5 5.3 4.5 15.4 4.0 7.8 9.0 5.5 3.1 4.9 5.7 1.4 6.4

8.7 1.1 4.1 12.2 5.8 5.0 14.1 5.3 8.6 9.0 5.3 1.5 4.7 6.9 1.8 6.2

10.3 1.2 5.1 12.1

8.6

1.1
4.0 10.6 4.3 5.1 15.8 5.7 6.7 10.3 5.8 3.0 4.4 7.3 2.0 4.6

5.1
3.8 14.3 4.2 7.3 8.6 5.6 3.3 4.7 6.3 1.4 5.8

the LMP material removedwasreducedand carboxymethylated

(32), resulting in a simpler gel electrophoresis pattern (see Fig.

myosin in 5 M guanidine hydrochloride and included, therefore, a contribution from the released LMP. Plots of the logarithm of the concentration against the square of the radius derived from

low speed sedimentationequilibrium studies(14) showed a slight

3). The LMP removedby DTNB exhibits a singlestrong band

and several minor bands, whereas the residual LMP released from

downward curvature indicative of a nonideal behavior and plots of l/M zD appagainst initial concentration were linear, yielding DTNB-treated myosin by incubating this material at TOOTtem- M, 197,000at infinite dilution. The more recent high speed

peratureovernight in the presence of 2 M urea exhibits an inverse pattern with two major bands(Fig. 3, b and d) in positionscorresponding to the minor bandsof Fig. 3, a and c, and a minor band in a position corresponding to the major band of Fig. 3, a and c. Thus, this finding suggests that DTNB effects the selective removal of onemajor componentof the LMP fraction amountingto 40 to 50% of the total LMP mass. This result is in substantial agreementwith recent studiesof Weeds (33), who has provided supportingevidencefrom radioautographs and primary structure studies of the DTNB and residual LMP fractions. Chymotryptic peptide maps of the labeled, carboxymethylated DTNB and residual LMP fractions indicate that thesefractions correspondto different proteins.

(Yphantis type) equilibrium experimentsof Dreizen et al. (3) strongly emphasize the presence of LMP in the 5 M guanidine hydrochloride-myosin system in that plots of log c against r2 exhibit a striking upward curvature in the low concentration region. In thesestudies,M, of the high molecularweight polypeptide componentwasestimatedby correcting for the assumed distribution of LMP throughout the liquid column. This technique is open to question becauseof the nonideal behavior of myosin chainsin 5 M guanidinehydrochloride and the apparent heterogeneity of LMP. We have therefore redetermined the molecularweight of the dissociated chainsin the absence of LMP and have utilized a moredetailedanalysisof the mass distribution through the cell than hasbeenusedin previous investigationsin

20

Suhnit

Structure of Myosin
TABLE

Vol. 245, No. 1

V

Molecular

weights

of myosin
guanidine

subunit

free of small
Molecular

protein,

in 6 M

hydrochloride
weight5

Experiment

I CONC

2 (mg/ml)

FIG. 4. Concentration dependence of the reciprocal molecular weight averages of LMP-free myosin in 5 M guanidine hydrochloride. High speed sedimentation equilibrium (21,950 rpm). Molecular weight extrapolates to 193,000 g per mole at infinite dilution.

High speed 1 2 3 4 5 6 Average Low speed 1 3 5 7 8 9 10 Aver age

(Yphantis) 185,000 192,000 198,000 185,000 190,000 185,000 189,000 f 5,300 (Labar) 184,000 211,000 198,000 192,000 196,000 207,000 176, OOOb 198,000 f 9,800 at c = 0, for hydrochloride, each experiment, not included f2,500. in average.

M, = M, = M. b In 7 M guanidine

I CONC

2 hg/mll

FIG. 5. Concentration dependence of the reciprocal molecular weight averagesof LMP-free myosin in 5 M guanidine hydrochloride. Low speed sedimentation equilibrium (9,928 r-pm). Meniscus was depleted at 24,000 rpm. Molecular weight extrapolatesto 193,000 at infinite dilution.

order to assess the degreeof size heterogeneity among the dissociatedchains. The sulfhydryl groupsof myosin wereblocked by reaction with N-ethylmaleimide, and the product waspassed through a Sephadex G-150column (4 x 120cm) equilibratedwith 0.5 M KCl-0.01 M EDTA3 M urea at pH 7.0. The eluted myosin was precipitated from solution by dialysis against dilute KCI, and samples were then dialyzed against a pH 11.5 buffer and sedimentedat 52,000rpm in 30-mm optical path length cells to check for residualLMP. Under theseconditions,LMP could be detectedin the schlierenpattern at concentrations of lessthan 1% of the total protein. Only samples showingno trace of LMP wereused for equilibrium sedimentation studiesin 5 M guaniclme hydrochloride. Figs. 4 and 5 presenttypical resultsof equilibrium sedimentation at high and low speed. The resultsof each experiment are presentedas a plot of the reciprocal of the apparent molecular weight (l/M%, l/M,, and l/M,) as a function of concentration in the centrifuge cell, determined according to the procedure described under Materials and Methods.

Two featuresof interest emergefrom inspectionof Figs. 4 and 5. (a) The plots of l/M,,, against c are not linear, but exhibit an upward curvature which becomes more pronouncedin the higher averages. The reasons for this behavior are not clear, but it is possible that virial coefficientshigher than the secondorder coefficient are significant in the myosind M guanidinehydrochloride system. (b) In the low speedexperiments,the three curves (l/Mn, l/M,, l/M* plotted against concentrations)extrapolate to near coincidence at infinite dilution, indicating a high degree of homogeneitywith respectto sizeamongthe dissociated chains. Table V summarizes the molecularweightsobtainedfrom both high and low speed equilibrium sedimentationexperiments. Although the high speedstudiesgive a slightly lower value for the molecular weight of the dissociatedchains than the low speed experiments, this difference is still within experimental error. Although this differencein the molecularweightscould be caused by the presenceof a small amount of contaminating protein (0.5%) with a molecularweight of about 150,000 g per mole, no independentevidencefor the presence of sucha contaminant was found. The apparent specific volume (@) used in thesecalculations was obtained from triplicate density measurements (at 20) on two different LMP-free myosin preparations dialyzed at 20 against 5 M guanidine hydrochloride. Protein concentrations were 1.8 and 2.4%, respectively. Results yield @ = 0.710 f 0.005ml per g; i.e. the samevalue as that usedin the earlier calculationsof Woods et al. (14) for the M, of myosinin 5 M guanidine hydrochloride. The average molecularweight from both high and low speed experimentsis 194,000,a value in good agreementwith the previously publishedvalue of 197,000(14) which wasdeterminedat low speedon a myosind M guanidinehydrochloride systemcontaining low molecularweight protein. The reasonfor this close agreement can be seenfrom an inspectionof Figs. 6 and 7, which presentresultsderived from both high and low speed studiesof a myosin-5 M guanidine hydrochloride system in the presence of about 9 y. LMP (i.e. nonfractionated myosin). Fig. 6 showsthe

Issue of January

10, 1970

J. Gazith, S. Himmelfarb,

and W. F. Hawington

21

CONC (mg /ml)

6. Concentration dependence of the reciprocal molecular weight averages of myosin which contained LMP in 5 M guanidine hydrochloride. High speed sedimentation equilibrium (22,000 mm).
FIG. I 2 I I I

In Fig. 7, the reciprocal weight average molecular weight is plotted against concentration .for myosin containing low molecular weight protein, at both high.and low speed. The high speed experiment clearly shows the presence of LMP. Attempts to extrapolate at the low concentration region to obtain the molecular weight of LMP (3) would give a much higher molecular weight for this component than any reported in the literature. Extrapolation from the high concentration region to obtain the molecular weight of the heavy component is also likely to yield an incorrect value, although the error would be less than in the first case. The high speed equilibrium sedimentation method is very sensitive to the presence of very small amounts of lower molecular weight species. In the particular case of the myosin subunit and LMP in 5 M guanidine hydrochloride, amounts of the latter corresponding to 0.5% or less of the total protein would be easily detected, especially when the x-average molecular weight is used. On the other hand, it can be seen from Fig. 7 that at low speed the results are unaffected by LMP and its presence would be undetected in the sample. This method would be more sensitive to the presence of aggregated protein. Thus, it is clear that the combined use of the number, weight, and z-average molecular weights increase the sensitivity and reliability of both the high and low speed methods. With the meniscus depletion method, extrapolation of the three molecular weight averages to zero concentration would clearly show the presence of a small amount of a low molecular weight contaminant, although its presence would be completely obscured had only the weight average molecular weight been used. In the low speed method, the molecular weights are extrapolated over a considerable concentration range, and this extrapolation is made more reliable by the fact that all three averages are used.
DISCUSSION

I I

I 2 CONC. (mg/ml)

I 3

I 4

7. Concentration dependence of the reciprocal weight average molecular weight for myosin which contained LMP at high speed sedimentation equilibrium (22,000 rpm) and at low speed equilibrium (9,000 rpm).
FIG.

results for high speed equilibrium. As would be expected, the presence of LMP has a marked effect on the number average molecular weight throughout the cell whereas the effect on the z-average molecular weight is smaller and limited to the low protein concentration region, where the small protein contributes a significant part of the total concentration. It is clear that attempts to extrapolate the number or the weight average molecular weights to obtain the molecular weight of the heavy protein (3) are arbitrary and can be misleading. Although the z-average molecular weight can be extrapolated in this case to yield a reasonably accurate value for the major component, this average is very sensitive to the presence of high molecular weight material, and small amounts of aggregated protein can easily lead to erroneous conclusions.

In this study, we have examined the effect of selective dissociation of low molecular weight protein on the enzymatic activity, a property which we would expect to be a sensitive indicator of conformational alterations within the globular region of myosin. This segment of the myosin molecule is now known to contain the sites of ATPase activity, and recent reports have shown that it is the focus for binding of LMP as well (7-9, 11, 12). On the basis of evidence presented under Results, it appears that 40 to 50% of LMP is nonessential for ATPase activity and the gel electrophoresis results indicate that this fraction represents one of the three major bands of the whole LMP mixture. The LMP residue fraction remaining bound to myosin after the DTNB treatment yields two major bands on gel electrophoresis, and Weeds (31) has shown that chymotryptic peptides from the 14C-labeled carboxymethylated protein of this fraction have a single thiol sequence and are therefore evidently derived from a single protein species. The question as to whether this LMP component is a true subunit of the myosin molecule is difficult to establish categorically, but it seems clear from this work that removal of this protein results in total loss of ATPase activity. Previous sedimentation, viscosity, and optical rotation studies (30) have provided evidence that the polypeptide chains of rabbit myosin are completely unfolded and dissociated in 5 M guanidine hydrochloride and the present results, showing the identity of M,, Mw, and M, at infinite dilution, support this conclusion. We are disinclined to place as much weight on the sedimentation equilibrium studies of myosin in 7 M guanidine hydrochloride as on those in 5 M guanidine hydrochloride, since the (1 - #p) term

22

Xubunit Structure

of Myosin

Vol. 245, No. 1

in the molecular weight equation becomes markedly sensitive to small errors in the apparent specific volume, #J, and the density of the solvent at high concentrations of salt. However, the low value of 176,000 g per mole estimated in 7 M guanidine hydrochloride is very close to the value (172,000) found in earlier low speed equilibrium studies in this solvent (14). There is now sufficient experimental and theoretical information at hand to provide confidence in the accuracy of subunit molecular weight determinations in aqueous guanidine hydrochloride if the appropriate precautions are taken in these measurements. For the myosin structure, these measurements assume special significance in defining a fundamental parameter of the molecule because it has proven extremely difficult to develop a consistent subunit structure from physical chemical studies of the parent molecule in solution. The reasons for the confusion result from a combination of factors including the high molecular weight and asymmetry, the presence of strongly bound and heterogeneous low molecular weight protein, and the marked tendency of myosin to undergo self-association reactions. Two recent developments have helped to clarify the situation. (a) The electron microscope study of Slayter and Lowey (34) has provided strong evidence that the basic architecture of the molecule is that of a two-stranded rather than a three-stranded (13) coiled-coil of polypeptide chains; and (a) the finding that myosin exists in a rapidly reversible monomer $ dimer equilibrium in high salt (0.5 M KCI-0.2 M Pod, pH 7) which is overshadowed by an unusually large nonideality (virial coefficient) term at protein concentrations above about 0.05% (25, 35). Thus, although molecular weights (low speed equilibrium sedimentation) extrapolated from data obtained above about 1 mg per ml are speciously high (590,000 g per mole), plots of three reciprocal averages, l/Mn, 1/M,, and l/M,, against protein concentration (high speed equilibrium) over the range 0.1 to 0.5 mg per ml exhibit a striking minimum near 0.3 mg per ml and converge at infinite dilutionTnear:460,000 g per mole. If we assume 9 f 2% LMP associated with myosin and 194,000 f 10,000 g per mole as the most probable weight of the major subunit, the molecular weight of the native protein-LMP complex should be 430,000 & 30,000 for a two-stranded molecule. This is reasonably good agreement considering the problem of estimating the amount of LMP accurately. As noted earlier, estimates by other authors range between 5 and 15%. Our results are not inconsistent with the concept that one component of low molecular weight protein is an integral and essential part of the molecule, but the stoichiometric relationship between this component and the heavy chains remains to be established as does the complete regeneration of ATPase activity in mixing experiments of heavy chain and this light chain component. We believe the protein released by DTNB treatment is most likely a contaminant, but it may well play some essential
role in the contractile process.

allowing us to read his manuscript prior to publication and to Dr. David Yphantis for the use of his computer program. REFERENCES
1. TSAO, T. C., Biochim. Biophys. Acta, 11,368 (1953). 2. KOMINZ, D. R., CARROL, W. R., SMITH, E. N., AND MITCHELL, E. R., Arch. Biochem. Biophys., 79, 191 (1959). 3. DREIZEN, P., HARTSHORNE, D. J., AND STRACHER, A., J. Biol. Chem., 241, 443 (1966). 4. OPPENHEIMER, H., BARANY, K., HAMOIR, G., AND FENTON, J., Arch. Biochem. Biophys., 116, 233 (1966). 5. LOCKER, R. H., AND HAGYARD, C. J., Arch. Biochem. Biophys., 120, 454 (1967). 6. OPPENHEIMER, H., BARANY, K., HAMOIR, G., AND FENTON, J., Arch. Biochem. Biophys., 120, 108 (1967). 7. GERSHMAN, L. C., DREIZEN, P., AND STRACHER, A., Proc. Nat. Acad. Sci. U. S. A., 66,966 (1966). 8. LOCKER, R. H., AND HAGYARD, C. J., Arch. Biochem. Biophys., 120, 241 (1967). 9. GAETJENS, E., BARANY, K., BAILIN, G., OPPENHEIMER, H., AND BARANY, M., Arch. Biochem. Biophys., 123, 82 (1968). 10. FREDERICKSON, D. W., AND HOLTZER, A., Biochemistry, 7, 3935 (1968). 11. D~EI&N, P., GERSHMAN, L. C., TROTTA, P. P., AND STRACHER, A.. J. Gen. Phvsiol.. 60. 85 (1967). 12. STR~CHER, A., B&h&. Bioph&. Bei. Commun., 36, 519 (1969). 13. KI~LLEY, W. W., AND HARRINGTON, W. F., Biochim. Biophys. Acta, 41, 401 (1960). 14. WOODS, E. F., HIMMELFARB, S., AND HARRINGTON, W. F., J. Biol. Chem., 238, 2374 (1963). 15. GERSHMAN, L. C., STRACHER, A., AND DREIZEN, P., J. Biol. Chem., 244, 2726 (1969). W. W., AND BRADLEY, L. B., J. Biol. Chem.. I 218. 16. KIELLEY, 653 (1956). G. L., Arch. Biochem. Biophys., 82,70 (1959). 17. ELLMAN, 18. ANSON, M. L., J. Gen. Physiol., 24, 399 (1941). W. W., KALCKAR, H. M., AND BRADLEY, L. B., J. 19. KIELLEY, BioL Chem.. 219. 95 (1956). M.F., END ENG~ANDER, S. W., Biochemistry, 2, 39 20. GELLERT, (1963). H., J. Phys. Chem., 66, 427 21. CASASSA, E. F., AND EISENBERG, (1961). D. A., Biochwnistry, 3, 297 (1964). 22. YPHANTIS, D. E., AND YPHANTIS, D. A., Ann. N. Y. Acad. Sci., 23. ROARK, 164, 245 (1969). F. L., Proc. Nat. Acad. Sci. U. S. A., 64,31 (1965). 24. LABAR, J. F., Ph.D. thesis, Johns Hopkins University, 1969. 25. GODFREY, T., AND KIELLEY, W. W., Biochim. Biophys. Acta, 81, 26. SEKINE, 336 (1964). T., BARNETT, L. M., AND KIELLEY, W. W., J. Biol. 27. SEKINE, Chem., 237, 2769 (1962). A., J. Biol. Chem., 239, 1118 (1964). 28. STRACHER, A., J. Biol. Chem., 236, 2467 (1961). 29. STRACHER, D. M., HARRINGTON, W. F., AND KIELLEY, W. W., 30. YOUNG, J. Biol. Chem., 237,3116 (1962). 223, 1362 (1969). 31. WEEDS, A., Nature, AND N. 0. KAPLAN (Edi32. HIRS, C. H. W., in S. P. COLOWICK tors), Methods in enzymology, Vol. 11, Academic Press, New York, 1967, p. 202. REISFELD, R. A., WYCKOFF, M., AND ZACCARI, 33. CHRAMBACH,.A., J.. Anal. Biochem.. 20,154 (1967). H. S., ANDLOWEY, s., Pkoc. Nat. Acad. Sci. U. S. A., 34. SLA&ER, 68, 1611 (1967). J. F., AND HARRINGTON, W. F., Biochemistry, in 35. GODFREY, press.

Acknowledgment-We

are grateful to Dr. A. G. Weeds for