j. biol. chem.-1970-gazith-15-22

8/12/2019 J. Biol. Chem.-1970-Gazith-15-22

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THE JOURNAL OF BIOLOGICAL CHEMISTR YVol. 245, No. 1, Issue of January 10, PP. 15-22, 1970

Printed in U.S .A.

Studies on the Subunit Structure of Myosin*

(Received for publication, October 31, 1968)

JOSEPH GAZITH,~ SYLVIA HIMMELFARB, AND WILLIAM F. HARRINGTON

From the Department of Biology, McCollum-Pratt Institute, the Johns Hopkins University, Baltimore, Maryland21218

SUMMARY

The low molecular weight protein associated with rabbitskeletal myosin has been estimated by gel titration studiesin 5 M guanidine hydrochloride and was found to constitute

9 f 2 of the myosin mass. We have found that 40 to50 of the low molecular weight protein can be removedfrom myosin without any apparent changes n the adenosine-triphosphatase activity. Attempts to remove a larger fractionof low molecular weight protein resulted in a concomitantloss of enzymatic activity.

The molecular weight of the major myosin subunit freedof low molecular weight protein has been redetermined in5 M guanidine hydrochloride and was found to be 194,000 gper mole. The significance of its presence n assessing hemolecular weight of the major subunit at low speed and highspeed sedimentation equilibrium has been investigated.

The presence of low molecular weight protein, or lightchains, 1 n myosin preparations has been clearly established ya variety of techniques n recent years (l-5). LMPl isolated byvarious methods has been shown o be heterogeneous y poly-acrylamide and cellulose acetate gel electrophoresis 6-8) andcolumn chromatography (9), and the weight fraction of LMPassociated ith myosin has been eported to be between 5 (9)and 15 (2), with molecular weights ranging between 20,000(7, 8) and 32,000 10) on the unfractionated LMP mixture. Onthe basis of the finding that LMP has an unusually strong affini tyfor myosin, and that it occurs n all myosin preparations, t hasbeen postulated as a subunit of the molecule 3, 5, 7, 11). Thisproposal has been given strong support by the recent studies ofStracher (12), who has shown hat rapid separation of low molec-ular weight protein from the heavy chains of myosin on SephadexG-200 columns equilibrated with 4 M LiCl, followed by remixingand dialysis, esults n about 30 recovery of the original ATPaseactivity of native myosin. Similar experiments with Subfrag-

* This work is Communication 574 from the Department ofBiology, the McCollum-Pratt Institute, The Johns HopkinsUniversi ty, Baltimore, Maryland, and was supported by UnitedStates Public Health Service Grant AM-04349.

$ Present address, Department of Microbiology, Bar Ilan Uni-versity, Ramat Gan, Israel.

1 The abbreviations used are: LMP, low molecular weight pro-tein; DTNB, 5,5-dithiobis(2-nitrobenzoic acid).

ment 1 yielded a recovery of 42y0. Moreover, the actin-bindingcapacity of these reconstituted systems seemed o follow theATPase activity.

In this work, we have attempted to dissociate ow molecular

weight protein selectively from myosin under conditions whichwould retain the enzymatic activity of the native protein. Aswe will show, only a fraction of the LMP mixture (about 50y0)is required for full ATPase activity. We have also determinedthe molecular weight of LMP-free myosin in 5 M guanidmehydrochloride because revious determinations 3, 7, 11, 13, 14)of the size of the major subunit in this solvent have been carriedout on the myosin-LMP mixture.2

MATERIALS AND METHOD S

Myosin was prepared by the method of Kielley and Bradley(16) unless otherwise noted and included, as a final step, am-monium sulfate fractionation between 40 and 50 saturation.

For resolution of LMP and myosin by gel filtration, SephadexG-100 gel was equilibrated with 5 M guanidine hydrochloride andpoured into a column (2.5 x 100 cm) which was adapted forreverse low to prevent packing of the gel. Myosin was allowedto react with a lo-fold molar excess with respect o -SH groups)of N-ethylmaleimide overnight at 5 (13), and the resultingsolution, made 5 M with respect o guanidine hydrochloride, wasapplied to the column at a concentration of less han 0.5 .Higher protein concentrations esulted n poor resolution of theelution patterns because f the high viscosity of the sample.

For reversible blocking of -SH groups, myosin was allowedto react with 0.01 M DTNB or p-chloromercuribenzoateat pH 8.5 in 0.5 M KCI-0.01 M Tris-HCI. When DTNB was

used, 0.01M

EDTA was always present. In some cases, he re-action was carried out in the presence f 1 M or 2 M urea. Re-moval of the dissociated MP was achieved by dilution-precipi-tation of the myosin at an ionic strength of 0.05. The blockingreagent was emoved and the sulfhydryl groups were regeneratedby dialysis against 0.5 M KCl, 0.01 M EDTA, and 0.01 M /3-mercaptoethanol or dithiothreitol, pH 7.0 to 8.5. The extentof sulfhydryl regeneration was assayed with DTNB in 1 M gua-nidine hydrochloride-O.01 Tris-HCl-0.01 M EDTA, pH 8.5

2 Since this paper was submitted, two reports have appeared inwhich the molecular weight of the major subunit was determinedwith the use of LMP-free myosin in 5M guanidine hydrochloride.A value of 232,000 to 238,000 was estimated by Frederickson andHoltzer (10) from viscos ity measurements alone, and Gershmanet al. (15) obtained 212,000 using high speed sedimentation equilib-rium.

.5


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16 Subunit Xtructure of Myosin Vol. 245, No. 1

(17). Guanidine hydrochloride was used to accelerate the timerequired for complete reaction. Irreversible blocking of -SHgroups was accomplished by causing myosin to react with N-ethylmaleimide as described previously.

All chemicals were reagent grade except as noted. Guani-dine hydrochloride was prepared from guanidine carbonate by

the method of Anson (18) and dried in a vacuum desiccator overP&s. Prior to use, the guanidine hydrochloride was examinedfor the presence of ultraviolet-absorbing materials by comparinga solution of 7 M guanidine hydrochloride against water. Ureawas recrystallized from 70% ethanol solutions.

Calcium-activated ATPase activity of myosin was determinedas before (16); the activity in the presence of EDTA was esti-mated according to the method of Kielley, Kalckar, and Bradley(1%.

LMP-free myosin was prepared for molecular weight studiesby treating the native protein overnight at 5 with N-ethylmalei-mide, followed by gel filtration on a Sephadex G-150 column(4 x 120 cm) which had been previously equilibrated in 3M

urea-O.5M

KCI.The eluted LMP-free protein was adjusted to

5 M with respect to guanidine hydrochloride and dialyzed against5 M guanidine hydrochloride for 24 to 48 hours. The resultingmyosin solution was centrifuged at 50,000 rpm for 2 hours, andthe protein concentration was adjusted to that required forequilibrium sedimentation. This protein solution was redialyzedagainst 5 M guanidine hydrochloride for an additional 72 to96 hours and transferred directly to the centrifuge cell .

Protein concentrations of native myosin in 0.5M KC1 wereestimated from an extinction coefficient of 500 cm2 per g (atX = 280 mp) determined previously (20). The extinctioncoefficient of LMP-free myosin in 5M guanidine hydrochloride(535 cm2 per g at X = 280 rnpcl)was determined by the method ofKielley and Harrington (13). Solutions were prepared by weigh-ing the lyophilized protein into 5M guanidine hydrochloride.The amount of water bound to the protein was obtained bydrying triplicate samples under vacuum over PzOs at 110.

The apparent partial specific volume, $ (defined according toCasassa and Eisenberg (21)), of LMP-free myosin in 5M guani-dine hydrochloride was determined after 4 days of dialysis at20. Densities of protein solution and dialysate were measuredwith 25-ml pycnometers, and the protein concentration wasdetermined from measurement of the optical density (h = 280rnp) with the use of the extinction coefficient of LMP-free myosinin 5 M guanidine hydrochloride.

The extinction coefficient of LMP in 0.5M KC1 was determinedon material isolated from Sephadex G-150 columns equilibratedwith 5 M guanidine hydrochloride. Solutions were dialyzedexhaustively against water, and the protein concentration wasestimated from Kjeldahl analysis with the use of a nitrogen-pro-tein factor obtained from ammo acid analysis. The extinctioncoefficient of LMP in 5M guanidine hydrochloride was obtainedby comparing the optical density of a solution prepared by di-lution of an aliquot of LMP plus solid guanidine hydrochloridewith 0.5 M KC1 to an equivalent aliquot made up to the samevolume with 0.5M KC1 alone. Extinction coefficients for LMPin 0.5 M KC1 and 5 M guanidine hydrochloride were identical(500 cm2 per g at X = 276 mp).a

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

efficient for LMP (360 cm2 per g) than is reported in this paper(500 cm2 per g). If the lower extinction coefficient s used, theamount of LMP bound to myosin is raised to 12.5 f 2%.

Molecular weights of LMP-free myosin in 5 M guanidine hy-drochloride were estimated by both the high speed meniscusdepletion technique of Yphantis (22) (22,000 pm) and the lowspeed sedimentation equilibrium method (9,000 o 10,000 pm).Temperature was always 20. For selection of the appropriatespeed, he parameter a,,, = (M(1 - @p)w3/2 RT was used,

where p is the density of the solution and w is the angular veloc-ity. On the assumption hat M is 200,000 g per mole, he speedwas selected o that a, = 4.5 to 6.5 cm+ for high speed quilib-rium and 1.5 to 2.5 cmm2 or the low speed uns. Equilibriumwas established fter 24 to 48 hours, depending on the speed ndcolumn height, and was checked or both methods by comparingthe gradients on two plates taken at least 24 hours apart. Col-umn heights were 2 to 2.5 mm. Molecular weights were cal-culated as a function of concentration n the cell, by a computerprogram written by Roark and Yphantis (23).

After equilibrium was established and recorded in the lowspeed experiments, he speed was doubled and the decrease nconcentration near the meniscus was followed with time accord-

ing to a modification of the procedure of LaBar (24) (see Godfrey(25)), until complete depletion at this level of the cell occurred.To ensure complete depletion at the meniscus, he speed waselevated to 35,000 rpm at the end of the experiment, and theconcentration near the meniscus was ollowed further with time.In this way, the concentration near the meniscus ould be deter-mined to an accuracy of about 0.03 ringe and the concentrationat each point in the cell could be established. Although thismethod requires reading many photographic plates, it is inde-pendent of the assumption of conservation of mass. The sameprocedure was adopted n the high speed experiments o ensurecomplete depletion of the meniscus. Weight average molecularweights were calculated for each point in the cell by the equa-tion

(1)

where c is concentration and r is the distance rom the center ofrotation. The z-average molecular weight at each point in thecell is calculated in the Roark-Yphantis computer program bythe relation

and the number average molecular weight by the relation

M,(r) = 49

s de c(m)

-+-m Kh9 M,(m)

(2)

(3)

where c(m) and M,(m) are the concentration and number aver-age molecular weight at the meniscus.

The term (c(m))/(lll,(m)) in Equation 3 can be neglected nhigh speed experiments when c(m) is essentially zero. In lowspeed experiments, when c(m) is not equal to zero, the concen-tration at the meniscus s determined as mentioned above andthe problem s to choose a value for M,(m). This can be ap-proximated by a linear combination of M,(m) and M,(m), sothat (23)


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Issue of January 10, 1970 J. Gazith, X. Himmelfarb, and W. F. Harrington 17

MYOSIN

h0 j0.0 --.-.-.-..--.

Ld--. . .._ ~ _.____ j

I II II/I III I0 20 40 60 80 100 120 140 160 180 200 220 240

EFFLUEN T VOLUME (ml)

FIG. 1. Gel filtrat ion of myosin on a Sephadex G-100 columnin5 M guanidine hydrochloride. Applied, 8 ml of 0.5% myosin in5 M guanidine hydrochloride. The column was developed with aflow rate of 8 ml per hour. Fractions of 5 ml each were collected.Inset, to 43 mg of myosin in 5M guanidine hydrochloride, 5 mg ofribonuclease were added (Wort.hington, ethanol-fractionated).The mixed protein solution was applied to the column and elutedas above.

M&n) = t M&n) - + K(m) (4)

The number average molecular weight so obtained is an ap-proximation because of the assumptions involved in Equation 4;however, the error in M,(m) does not seem serious, becausevalues 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 withMyosin-Although low molecular weight material is not easilydetected in velocity sedimentation studies of myosin in thedissociating solvent 5 M guanidine hydrochloride, gel filtrationelution patterns in this solvent system clearly show the presenceof a minor component (LMP) trailing behind the major subnuitpeak. As can be seen from inspection of Fig. 1, this componentelutes ahead of ribonuclease, which suggests that the mo-lecular weight is probably greater than 14,000 g per mole. Forestimation of the relative amount of bound low molecular weightprotein released when the polypeptide chains of myosin areunfolded and dissociated in guanidine hydrochloride, the areaof each peak of the bimodal elution pattern was measuredand the concentration of each component was estimated fromits extinction coefficient (see Materials and Methods). Theaverage amount of low molecular weight protein dissociated frommyosin under these conditions approximated 9% of the totalprotein mass and seemed to be independent of the time of ex-traction or of the solvent conditions used in the isolation ofmyosin (Table I)?

For a quick, routine assay of the amount of bound low molec-ular weight protein, myosin was incubated in 2M urea (0.5 M-KCI-0.01 M EDTA) overnight at room temperature. This treat-ment also results in dissociation of LMP from myosin. Dilutionof the resulting solution with 2M urea to an ionic strength of0.05 precipitated the myosin, leaving LMP in the supernatantwhere its concentration could be determined by optical density

TABLE IAmount of small protein bound to myosin estimated by two methods

Small protein

Sample and method of myosin preparationa

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 ..................................

%

9.5 5.97.79.0 7.17.0

10.1 7.210.09.7 6.38.07.4 5.7

10.07.4 6.4

10.39.29.98.9 6.4

a A regular preparation is extraction of muscle mince in 0.5MKCl-0.1 M K2HP04, pH 9.3, extracted for 10 min unless otherwisenoted.

measurement. The amount of LMP associated with myosinestimated in this way was invariably lower than that determinedby gel filtrat ion (Table I), presumably as a result o f its coprecipi-tation with myosin. When the precipitated protein was redis-solved in pH 11.5 buffer (2) and examined at high speed in theultracentrifuge (see below), approximately 2 to 3% of the totalprotein was always present as LMP. Thus, these experiments,when taken in conjunction with the gel filtration studies, indi-cate that about 8 to 10% of LMP is strongly associated withthe native myosin molecule as judged by this criterion.

Effect of Varying Extraction Conditions on Amount of LowMolecular Weight Protein Associated with Myosin-The effect ofvarying the composition of the extraction medium on the amountof low molecular weight protein was also investigated in a series ofhigh speed ultracentrifugation experiments of myosin at pH 11.5in a solvent consisting of 0.4 M KCI-0.1 M Na&Oa. Underthese conditions, as was originally shown by Kominz et al. (2),LMP is released and is clearly resolved in the schlieren patternfrom the more rapidly sedimenting myosin peak. Measure-ments of the area of the LMP peak showed it to be directly pro-portional to the loading myosin concentration and, consequently,no correction was made for a Johnston-Ogston effect in estimatingthe concentration of this component. (Inasmuch as only rela-tive, not absolute concentrations are measured by this procedure,any error resulting from the neglect of the Johnston-Ogston cor-rection should be negligible.) The area of the LMP peak at pH11.5 was compared to that found in a standard (Kielley-Bradley)myosin preparation (assumed as 100% LMP) which was centri-fuged at the same time in a companion 12-mm double-sectorultracentrifuge cell. The compositions of the various extractionmedia are given in Table II. Myosin was also extracted frommyofibri ls prepared from the back muscle of rabbit and storedin 50% glycerol at -20 for over 1 year. These were washedexhaustively to remove soluble proteins and were used to extractmyosin by the Kielley-Bradley method. In all of these prep-


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18 Subunit Structure of Myosin Vol. 245, No. 1

TABLE II

Compositions f various solvents used for extracting myosin

KC1KpHP04KC1KzHPOdKzHPO4KC1&CO,KHCOsKzHPOdKaHP04

Af

10.1 9.320.1 9.30.226 9.30.60.010.04 9.30.226 10.80.226 6.5

arations, he amount of LMP released rom myosin was constantand independent of the extraction medium composition.

The apparent invariance in the amount of low molecularweight protein associated with myosin isolated n the presenceof a wide variety of extracting solvents suggests, n agreementwith the conclusions f earlier workers, that this material maybe an essential omponent of the myosin structure.

Effect of Removal of Low Molecular Weight Protein on ATPaseActivity----The methods used so far for the removal of LMP alsoresult in denaturation of the myosin molecule, and a search wasinitiated for relatively mild conditions hat would dissociate owmolecular weight material n the absence f significant alterationsin ATPase activity. Because he ATPase activity of myosin sknown to be ntimately dependent n specific SH groups withinthe globular region of the molecule 26-28), a general procedurewas developed based on reaction of all of the thiol groups of theprotein with a removable blocking reagent. LMP is then re-leased rom myosin n the presence f a dissociating olvent (seebelow), and the residual myosin is precipitated at low ionicstrength. The blocking groups are then removed by dialysisagainst @-mercaptoethanol r dithiothreitol and the resultingmyosin examined or ATPase activity and residual LMP.

Reaction of the sulfhydryl groups of myosin with the remov-able blocking reagents p-chloromercuribeneoate r DTNB aloneresults n a bimodal schlieren pattern in the ultracentrifuge athigh speed. The slow component showed edimentation harac-teristics similar to those of the LMP peak observed at pH 11.5.No loss n ATPase activity was observed after precipitation ofthe myosin ollowed by regeneration f the -SH groups. Cen-trifugation in alkali indicated the removal of 15 to 20 LMP.

For further enhancement f the dissociation f low molecularweight protein, myosin was allowed o react with DTNB in thepresence f varying concentrations of urea. Urea was used asthe dissociating olvent of choice because t has been previouslyshown to be a rather mild denaturing agent for myosin at lowconcentrations 29). We found incubation of myosin or 15 minat 4 in 1 M urea-O.01 DTNB to be the optimum conditions ordissociation f LMP and regeneration f ATPase activity (Fig. 2).About 40 to 50 of LMP was released nder these conditions,the major fraction of which could be removed by precipitation ofthe myosin with no apparent oss n either Ca++- or EDTA-acti-vated ATPase activity, or in the number of regenerated hiolgroups (Table III). If the sulfhydryl groups are regeneratedwithout removing LMP (i.e. in the absence f the precipitationstep), the released ow molecular weight material reassociates

FIG. 2 (upper). Ultracentrifuge pattern of LMP released fterre-action of myosin with DTNB in the presence f urea. To 1.6a/, myo-sin solution, DTNB was added o a final concentration of 0.01 Mand urea to a final concentration of 1M. DTNB and urea weresimilarly added to the buffer . A filledEpon double-sector ellwas used at 52,000 pm. Temperature, 3; bar angle, 70. Con-vection spikes are the result of the high myosin concentration nthis experiment.

FIG. 3 (lower). Disc gel electrophoresis of reduced, carboxymeth-ylated LMPfractions in7.5 polyacrylamide,pH9.3. a, LMP frac-tion removed by treatment with DIWB-2 M urea at 4. Stainedwith Amido schwarz dye. b, LMP residual fraction removed bytreatment of the myosin from a with 2 M urea overnight at roomtemperature. Stained with Amido schwarz dye. c, same as a,stained with Coomassie brilliant blue (33). d, same ss6, stainedwith Coomassie brilliant blue.

with myosin and subsequent elocity sedimentation tudies showonly a single, hypersharp schlieren eak. Attempts to retain fullATPase activity while dissociating a larger fraction of LMP inthe presence f higher concentrations of urea have been unsuc-cessful n our hands. Attempts to prevent loss of activity byincluding ATP, Pod-, or Ca++ n the dissociating medium werealso unsuccessful. In 1.5 M or 2 M urea (0.01 M DTNB), 70 to80 of the LMP was removed from myosin, but with a con-comitant 70 to 90 loss n ATPase activity. Dissociation ofLMP could be reversed by removing the urea and regeneratingthe sulfhydryl groups as before, but the depression n ATPaseactivity was not reversed Table III). Following reaction withthe thiol-blocking reagents n 2 M urea, only a fraction of thesegroups could be regenerated, n agreement with earlier studies


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Issue of January lo ,1970 J. Guzith, S. Himmelfarb, and W. F. Hawington 19

TABLE III TABLE IVEff ect of removal of small protein on enzymatic activ ity oj myosin Amino acid compositionof low molecular weight protein isolated

by different methods

Preparation

Urea, 1 M, plus DTNB, 0.01 M1

23

4Control

Urea, 2 M, plus DTNB, O.Ol M1356Control

SIUallprotein

remaining

61 0.8559 1.0068 0.80

59 1.0054 1.0058 1.00

100 1.00

4446

100

0.10 6.00.31 5.30.0 4.20.0 4.50.15 4.5

R&tiWactiviv

julfhydryl:roups per

106 g ofmywin

6.3

5.98.56.3

7.3

7.2

Q Following removal of DTNB from myosin by dialysis against0.5 M KCl-0.01 M EDTA and 0.01 , mercaptoethanol r dithio-threitol. Myosin in 0.5 M KC1 was ncubated for 15 min in thepresence f the dissociating system at 4, then precipitated fromsolution by lowering the ionic strength to 0.05 M KC1 either byadding 2 M urea or water.

(30) in which attempts were made o regenerate myosin ATPaseactivity from the fully dissociated and unfolded polypeptidechains n 5 M guanidine hydrochloride.

Because nly about 40 to 50 of low molecular weight proteincan be removed rom myosin without loss of ATPase activity, itis important to establish whether the DTNB treatment effects aselective emoval of one component or dissociates he whole LMPmixture to varying degrees. Amino acid analysis of LMP iso-lated after reaction with DTNB alone (15 to 25 dissociation),DTNB in the presence of 1 to 2M urea (4; 40 to 80% dissocia-tion), or LMP isolated by column chromatography in the pres-ence of 5 M guanidine hydrochloride showed no sign&ant trends(Table IV), and all showed igh phenylalanine o tyrosine ratiosas eported earlier (5, 9). Disc electrophoresis of these prepara-tions showed a pattern consisting of three major bands andseveral minor bands. According to a suggestion of Weeds (31),the LMP material removed was educed and carboxymethylated(32), resulting in a simpler gel electrophoresis pattern (see Fig.3). The LMP removed by DTNB exhibits a single strong bandand several minor bands, whereas the residual LMP released fromDTNB-treated myosin by incubating this material at TOOT em-perature overnight in the presence f 2 M urea exhibits an inversepattern with two major bands Fig. 3, b and d) in positions corre-sponding o the minor bands of Fig. 3, a and c, and a minor bandin a position corresponding to the major band of Fig. 3,a and c.Thus, this finding suggests hat DTNB effects the selective re-moval of one major component of the LMP fraction amounting o40 to 50 of the total LMP mass. This result is in substantialagreement with recent studies of Weeds (33), who has providedsupporting evidence rom radioautographs nd primary structurestudies of the DTNB and residual LMP fractions. Chymo-tryptic peptide maps of the labeled, carboxymethylated DTNBand residual LMP fractions indicate that these ractions corre-spond o different proteins.

All analyses were on duplicate samples hydrolyzed at110 for24 hours. No corrections were made for time-dependent losses.

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

8.30.7

3.013.6

5.33.5

16.04.0

7.49.46.3

3.84.86.3

1.66.1

cysteine. . . . .

9.31.2

3.511.4

4.64.4

15.3

5.2

7.59.5

6.03.14.76.2

1.75.2

11.9

0.83.9

13.2

5.53.6

13.9

3.9

7.88.95.33.3

4.75.61.36.5

10.10.94.0

12.5

5.34.5

15.4

4.0

7.89.05.53.1

4.95.71.4

6.4

8.71.14.1

12.25.85.0

14.15.3

8.69.05.31.5

4.76.91.8

6.2

10.3 8.61.2 1.15.1 4.0

12.1 10.65.1 4.33.8 5.1

14.3 15.84.2 5.7

7.3 6.78.6 10.35.6 5.83.3 3.04.7 4.46.3 7.31.4 2.05.8 4.6

1.0 0.6- -

= % LMP removed.b Sample reduced and carboxymethylated before analysis (see

Reference 31).

I I I IU2, C &r- DTNB DTNB(col- DTNB omerc- +1x +2x(15-25) uriben- urea urea

mate (40-45) (70-80)(15-25)

0 Residual is LMP remaining after 2M urea-DTNB treatment

and is removed by treatment at room temperature overnight inthe presence of 2 M urea.

Urea,2d

(resid-IlaP)

Molecular Weight of Constituent Poplypeptide Chains of Myosin-The weight average molecular weight (M,) of the dissociatedpolypeptide chains of myosin has been reported in earlier Archi-bald and sedimentation equilibrium studies (13, 14). Theseexperiments were performed on solutions of unfractionatedmyosin in 5 M guanidine hydrochloride and included, therefore, acontribution from the released LMP. Plots of the logarithm ofthe concentration against the square of the radius derived fromlow speed edimentation equilibrium studies 14) showed slightdownward curvature indicative of a nonideal behavior and plotsof l/M zD pp against initial concentration were linear, yieldingM, 197,000 at infinite dilution. The more recent high speed(Yphantis type) equilibrium experiments of Dreizen et al. (3)strongly emphasize he presence f LMP in the 5 M guanidinehydrochloride-myosin system in that plots of log c against r2exhibit a striking upward curvature in the low concentrationregion. In these studies, M, of the high molecular weight poly-peptide component was estimated by correcting for the assumeddistribution of LMP throughout the liquid column. This tech-nique is open to question because of the nonideal behavior ofmyosin chains n 5 M guanidine hydrochloride and the apparentheterogeneity of LMP. We have therefore redetermined themolecular weight of the dissociated hains n the absence f LMPand have utilized a more detailed analysis of the mass istributionthrough the cell than has been used n previous nvestigations n


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20 Suhnit Structure of Myosin Vol. 245, No. 1

0 I 2 3 4

CONC (mg/ml)

FIG. 4. Concentration dependen ce of the reciproca l molecu larweight averages of LMP-free myosin in 5M guanidine hydro-chloride. High speed sedimentation equilibrium (21,950 rpm).Molecular weight extrapolates to 193,000 g per mole at infinitedilution.

0 I 2 3 4

CONC hg/mll

FIG. 5. Concentration dependen ce of the reciproca l molecu larweight averages f LMP-free myosin in 5M guanidine hydro-chloride. Low speed sedimentation equilibrium (9,928 r-pm).Meniscus was depleted at 24,000 rpm. Molecular weight extrap-olates o 193,000 at infinite dilution.

order to assess he degree of size heterogeneity among the dis-sociated chains.

The sulfhydryl groups of myosin were blocked by reaction withN-ethylmaleimide, and the product was passed hrough a Sepha-

dex G-150 column (4x

120 cm) equilibrated with 0.5M

KCl-0.01M EDTA3 M urea at pH 7.0. The eluted myosin was precipi-tated from solution by dialysis against dilute KCI, and sampleswere then dialyzed against a pH 11.5 buffer and sedimented at52,000 pm in 30-mm optical path length cells to check for re-sidual LMP. Under these conditions, LMP could be detected nthe schlieren pattern at concentrations of less han 1 of thetotal protein. Only samples howing no trace of LMP were usedfor equilibrium sedimentation tudies n 5 M guaniclme ydrochlo-ride.

Figs. 4 and 5 present ypical results of equilibrium sedimenta-tion at high and low speed. The results of each experiment arepresented as a plot of the reciprocal of the apparent molecularweight (l/M , l/M,, and l/M,) as a function of concentrationin the centrifuge cell, determined according to the proceduredescribed nder Materials and Methods.

TABLE V

Molecular weights of myosin subunit free of sma ll protein, in 6 Mguanidine hydrochloride

Experiment Molecular weight5

High speed (Yphantis)

1 185,0002 192,0003 198,0004 185,0005 190,0006 185,000Average 189,000 f 5,300

Low speed (Labar)1 184,0003 211,000

5 198,0007 192,0008 196,000

9 207,00010 176, OOObAver age 198,000 f 9,800

M, = M, = M. at c = 0, for each experiment, f2,500.b In 7 M guanid ine hydrochloride, not include d in average.

Two features of interest emerge rom inspection of Figs. 4 and5. (a) The plots of l/M,,, against c are not linear, but exhibitan upward curvature which becomes more pronounced n thehigher averages. The reasons or this behavior are not clear, butit is possible hat virial coefficients higher than the second ordercoefficient are significant n the myosind M guanidine hydrochlo-ride system. (b) In the low speed experiments, he three curves(l/Mn, l/M,, l/M* plotted against concentrations) extrapolate

to near coincidence t infinite dilution, indicating a high degreeof homogeneity with respect o size among he dissociated hains.Table V summarizes he molecular weights obtained rom both

high and ow speed quilibrium sedimentation experiments. Al-though the high speed studies give a slightly lower value for themolecular weight of the dissociated chains than the low speedexperiments, this difference is still within experimental error.Although this difference n the molecular weights could be causedby the presence of a small amount of contaminating protein(0.5 ) with a molecular weight of about 150,000 per mole, noindependent evidence or the presence f such a contaminant wasfound.

The apparent specific volume (@) used n these calculationswas obtained from triplicate density measurements at 20) ontwo different LMP-free myosin preparations dialyzed at 20against 5 M guanidine hydrochloride. Protein concentrationswere 1.8 and 2.4 , respectively. Results yield @ = 0.710 f0.005 ml per g; i.e. the same value as that used n the earlier cal-culations of Woods et al. (14) for the M, of myosin n 5 M guani-dine hydrochloride.

The average molecular weight from both high and low speedexperiments s 194,000, a value in good agreement with the pre-viously published value of 197,000 14) which was determined atlow speed on a myosind M guanidine hydrochloride system con-taining low molecular weight protein. The reason or this closeagreement an be seen rom an inspection of Figs. 6 and 7, whichpresent esults derived from both high and low speed tudies of amyosin-5 M guanidine hydrochloride system in the presence fabout 9 y. LMP (i.e. nonfractionated myosin). Fig. 6 shows he


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Issue of January 10, 1970 J. Gazith, S. Himmelfarb, and W. F. Hawington 21

CONC (m g /ml)

FIG. 6. Concentration dependence of the reciprocal molecularweight averages of myosin which contained LMP in 5M guanidinehydrochloride. High speed sedimentation equilibrium (22,000mm).

I 2 I I I

I I I I0 I 2 3 4

CONC. (mg/ml)

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

results for high speed equilibrium. As would be expected, thepresence of LMP has a marked effect on the number averagemolecular weight throughout the cell whereas the effect on thez-average molecular weight is smaller and limited to the low pro-tein concentration region, where the small protein contributes asignificant part of the total concentration. It is clear that at-tempts to extrapolate the number or the weight average molecularweights to obtain the molecular weight of the heavy protein (3)are arbitrary and can be misleading. Although the z-averagemolecular weight can be extrapolated in this case to yield areasonably accurate value for the major component, this averageis very sensitive to the presence of high molecular weight material,

and small amounts of aggregated protein can easily lead toerroneous conclusions.

In Fig. 7, the reciprocal weight average molecular weight isplotted against concentration .for myosin containing low molec-ular weight protein, at both high.and low speed. The high speedexperiment clearly shows the presence of LMP. Attempts toextrapolate at the low concentration region to obtain the molec-ular weight of LMP (3) would give a much higher molecularweight for this component than any reported in the literature.Extrapolation from the high concentration region to obtain themolecular weight of the heavy component is also likely to yieldan incorrect value, although the error would be less than in thefir st case. The high speed equilibrium sedimentation method isver y sensitive to the presence of ver y small amounts of lowermolecular weight species. In the particular case of the myosinsubunit and LMP in 5M guanidine hydrochloride, amounts ofthe latter corresponding to 0.5% or less of the total protein wouldbe easily detected, especially when the x-average molecular weightis used. On the other hand, it can be seen from Fig. 7 that atlow speed the results are unaffected by LMP and its presencewould be undetected in the sample. This method would be moresensitive to the presence of aggregated protein. Thus, i t is clearthat the combined use of the number, weight, and z-averagemolecular weights increase the sensitiv ity and reliability of boththe high and low speed methods. With the meniscus depletionmethod, extrapolation of the three molecular weight averagesto zero concentration would clearly show the presence of a smallamount of a low molecular weight contaminant, although itspresence would be completely obscured had only the weight av-erage molecular weight been used. In the low speed method, themolecular weights are extrapolated over a considerable concen-tration range, and this extrapolation is made more reliable by thefac t that all three averages are used.

DISCUSSION

In this study, we have examined the effec t of selective dissocia-tion of low molecular weight protein on the enzymatic act ivi ty,a property which we would expect to be a sensitive indicator ofconformational alterations within the globular region of myosin.This segment of the myosin molecule is now known to contain thesites of ATPase act ivi ty, and recent reports have shown that it isthe focus for binding of LMP as well (7-9, 11, 12). On the basisof evidence presented under Results, it appears that 40 to 50%of LMP is nonessential for ATPase act ivi ty and the gel electro-phoresis results indicate that this fraction represents one of thethree major bands of the whole LMP mixture. The LMP residuefraction remaining bound to myosin after the DTNB treatmentyields two major bands on gel electrophoresis, and Weeds (31) hasshown that chymotryptic peptides from the 14C-labeled carboxy-methylated protein of this fraction have a single thiol sequenceand are therefore evidently derived from a single protein species.The question as to whether this LMP component is a true subunitof the myosin molecule is difficu lt to establish categorically, butit seems clear rom this work that removal of this protein resultsin total loss of ATPase activ ity.

Previous sedimentation, viscosity , and optical rotation studies(30) have provided evidence that the polypeptide chains of rabbitmyosin are completely unfolded and dissociated in 5M guanidinehydrochloride and the present results, showing the identity ofM,, Mw, and M, at infinite dilution, support this conclusion. Weare disinclined to place as much weight on the sedimentationequilibrium studies of myosin in 7M guanidine hydrochloride ason those in 5 M guanidine hydrochloride, since the (1 - #p) term


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j. biol. chem.-1970-gazith-15-22

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