J. Biol. Chem.-1969-Gershman-2726-36

8/12/2019 J. Biol. Chem.-1969-Gershman-2726-36

1/11

Subunit Structure of MvosinJIII. A PROPOSID RIODEI, FOR RABBIT SIIIIIEOSIN*

(Received for publication, November 12, 19G8)LEWIS C. GERSHMAN,: X. STI~ACHEH,# ASI) I. DHEIZES~Prom the Departments of Medicine and Biochemistry, State Unirerhg of Xew Yodi Downstate Uedical Center,Brooklyn, New York 11203

SUMMARYHigh speed sedimentation equilibrium experimentsindicate a molecular weight of 468,000 (&lO,OOO) for rabbit

skeletal myosin in 0.4 M KCl-0.05 M phosphate, pH 6.5.Weight average molecular weight values of 520,000 or above,which are presumably due to aggregation, are obtained inultracentrifugal experiments on the same preparations ofmyosin. At pH 11, in 2 M guanidine, and on heat treatment,myosin is dissociated into a core of molecular weight 420,000weight and light subunits of average molecular weight20,200. The core may be dissociated by 5 M guanidine intotwo heavy subunits, of molecular weight 212,000 (=tS,OOO),having no evident COOH-terminal group on digestion withcarboxypeptidase A. The light alkali component comprises11.7% ( f 1%) of the protein, or 2.7 ( hO.3) light subunits permyosin molecule, for unchromatographed myosin andmyosin chromatographed once on diethylaminoethyl cellu-lose or cellulose phosphate; the molar ratio is reduced to 2.0(hO.3) in a small fraction of the original myosin that isrecovered after three chromatographic cycles on cellulosephosphate. Myosin may be reconstituted after subunitdissociation at alkaline pH or in guanidine solution; however,the reassociation of light subunits is diminished after pro-longed denaturation. The removal of light subunits isaccompanied by aggregation of the heavy chain core to lown-mers. In general, those denaturing conditions which leadto light subunit dissociation also result in the irreversibleinactivation of adenosine triphosphatase and, on heatdenaturation, values of AHf and AS: are comparable forboth processes. The ATPase activity of myosin may bepartially protected on alkaline treatment in 2 M KCl-0.01 MATP; enzymic activity is lost in identically treated but

* This stlidy was supported hv Rcscarch Grants U-13(X (toP.I>.) from th& Ilcalth it&arch C ouncil of the City of ?;ew Ydrkand GM-07076 (o A.S.) and ARI-001G.5 ( to P.11.) from the UnitedStates Public Health Service. This work wa s taken in part from athesis submitted by L.C.(;. for the dcgrcc o f Doctor of Philosophyin Phvsioloev and Bioohvsics. State Univcrsitv of Kcw York.Porlidns of zis work w&e pre.&nted at the 13thAnnual Meetingof the Biophysical Society (W, Gl). The preceding paper inthis series is Reference 2.$ hIcdic~ul Scientist Fel low of the Lift Tnsurrtnce Medical Re-search Fund.0 Career Scientist of the IIenlth Research Council of the Cityof Se\\- York.

fractionated heavy chain core and is augmented in thepresence of excess light subunits.

Previous st.udies (I-3) i11 t.his series indicate that at. pII IIabout three light or globular subunits of molecular weight 20,000may be dissocint.ed from the heavy meromyosin end of rabbitskeletal myosin, and i.he r&dual protein may bc dissociated b35 nr guanidine into two heavy or fibrous subunits of molecularweight 210,000 to 220,000. .\Iost recent experiments indicatea molecular weight of approximately 500,000 for rabbit skeletalmyosin (49). While appreciably higher xxlues (10, 11) ma>result from aggregation of myosiii (3, 5, 12), some uncertaintyremains nhrt.her the 500,000 ~11ue 111:q itpelf reflect minordegrees of aggregation and, in this respect., wight averagemolecular weights of 480,000 to 524,000 (4-7) do exceed ii singlenumbrr average value of 470,000 (i). Sirwe knowledge ofmonomer weight is important in the interpretation of subunit,comlwsit ion, the quest.ion has been explored by means of highspeed and low speed wdimentation equilibrium esperimentson identical preparations of myosin; prcliminay data have bernreported (2, 3).

A low molecular wcight component may be di*wriatcd frommyosin at alknlinc pH (2, 3, 13-IS), in 5 Y gunnidine (l-3, 13),in urea solutions (3, 13, 19, 20), in concentrated ealt solutionsat neutral 111 (21), and 011 heat crcat.ment (16,22, 23), succinyln-tion (24-26), and acetylation (16, 27). The light componentcomprises about 127; of unchrorllntogruphed rnyo&l (2, 3), hasa well defiurd amino acid comlmsition (14, 19, 26, 27), andhas COOH-terminal isolrucine (2, 14, 18, 22). The rondit,ionsleading to the diswciation of light component from nativemyor;in also result. in the inactivation of adcnosine triphosl)hatase,and attempts have been made at chromatographic separation oft.he light component from myosin preparations. Although thelight component is still precut. after chromatography on diethyl-aminoelhyl cellulose (27, 28) and DEAE-Scphades (17, IS), ithas been reported (26) t.hat myosiu contains only 4.576 of lo\vmolecular weight material aftclr combined chromatography (29)011 cellulose phosphate a11d I)EAE-cellulose. This nmbiguit>and reported differences in the proportion of light component(13-16, 27) prompted further stoi&iomctric studies on the alkali

2726


2/11

Issue of May 25, 1969 L. C. Gershman, A. Stracher, and P. Dreizen 2727components of unc:hromntographed and chromatographedmyosiii. Studies were also conducted on the time de1xndence ofdissociation and reassoci:Aon of light component with theheavy chain core, aud on possible protection of ATPase duringsuch treatment; preliminary findings have bec~l reported (21).

hZoleculnr weight dctrrminntions on the heavy suhunits(l-3, 10, 11, 17) and COOII-terminal analyses on myosin (30,31), heavy meromyosin (31), and Subfragmcnt 1 (32) have beenon preparations containing, or very likely containing, lightcomponent in molar 1)roportion or greater. Ultracentrifugalespcriment.s and COOI-I-terminal analyses on the purified heavysubunits arc here dcacribcd.

METIIODSRabbit. skeletal myosin, prepared essentially by the method of

Szent-Gybrgyi (1, 33), was stored in 0.4 M KC1 at pI1 6.5 untiluse. Some prc1)nrations were further chromat.ographed onDE.U -ccllulo~c (34) or celluloac phosphate (Signla) (26, 20) bydetailed proccclurci; most experiments were done 011 uiichroma-tUgraph(,d myosin.

The procedures used for ~1udies on myosin aL equilibrium in5 Y gu:midine and at pI1 11 have been described (1, 2). Inexperiments on the time tlcpendence of alkaline denaturation,the protein solution was t.itrated to alkaline pH with li,CO1-KOII solution, and reversibility was studied after back tit.ra-t.ion or dixlgsis to neutral pFI. For comparable espcrimcnts in1 M or 2 JI guanidine, concentrated guunidine-0.4 JI KC1 wasadded to prot,ein solut.ioll, and rever~ibi1it.y was studied afterdialysis against 0.4 M KCI-0.005 nr NalICOs, pII 7. IIeat-treated mywin was incubated in a water bath at 25-40 forperiods up to 24 hours.

The heavy subunits of myosin were purified by one of thefollowing methods: (a) alkaline fractionation (2), in which pro-tein was dialyzed agniust 0.4 11 IiCl-0.1 nr Sa2C03, 1)H II,titrated to pIi 7 with 1 M K&PO,, and precipitated on dilutionwith water, and (b) guanidine fractionation, in which the proteinwas treat.etl in 2 M or 5 M gunnidine.HCl-0.001 M EWJh forperiods up to 24 hours and xdtcd out with ammonium sulfate(35 to 45% fract,ion) or 0.8 M potassium citrate (26), and theprecipitate was wwh cd in dist.illed water. Two or three cycleswere performed for each 1)rocedure.

T~ltrnccnt.rifugal esperimcnts were done at, 4 in a Beckmanmodel E analytical ultraccntrifugr equipped wi th xchlirren andinterference optics and, for the later work, with electronic speedcontrol. III sedimentat.iou velocity experiments the prol)ortionsof light and heavy components were det.ermined from schlierenmeasurements corrected for radial dilution and JohnstowOgstoneffect (2, 35). IIigh speed sedimentation equilibrium esperi-merits w wc done by the method of Yphnntis (36); multicom-ponent analysis is based on data obtained at successive equilibriaat two or more rotor speeds, as clwribcd elsewhere (2, 37). Inthe experiments at pH 11, after heat treatment, and in 2 31guanidinc, equilibrium data were customarily obtained after 29to 40 hours at 13,000 t.o 14,000 rpm and af ter 14 to 24 hours at36,000 Lo 40,000 rpm. Corrections for Wiener skewing (36) werenegligible. I,:nv speed sedimentation equilibrium cy)erimentswere analyzed by the whole column method (38), and .\rchibnldmeasurements are based on combined schlicrewinterfercncemeasurwwnts (39). Absorption optics and a photoelect,ricw:tmwr were II& in several experiments that are dexribed

I I I I I0 5 IO 15 20concentration mg/ml

PIG. 1. Reciprocal of sedimentation coeficieut (SW-~) plottedagainst initial concentration of myosin, in 0.4 M KCI-0.05 11K&PO,, pH G.5, at 4.esplicitly. Equilibrium experiments were usually done with12-mm double sector durnluminum-filled or chorcoa-filled Eponcenterpieces; a sis-cornpnrt.lneiIt charcoal-filled Epnn centerpiecewas used in some of the low speed equilibrium espcriments.Synthetic boundary experiments were done wit-h twin capillarycent.erpieces, at. protein conccnt~rations of 3 to 6 mg per ml.

Protein conccntrat.ion was dctcrmincd from optical density at2800 A measured with a Zeiss NIQ II spectrophotometer mdcorrected for Rnyleigh scattering. Value for extinction coef f~-cicnts, refractive index increments, and partial specif ic volumeof myosin were as noted (1, 2). Values were assumed to beident.iwl for the purified heavy subunibs. The extinctioncoeffXcnt for the light component was determined as A&,, 3.5,based on Kjeldnhl analysis; aawning 16.7yc nit.rogen.

Ca++-activated ATPaw was determined on samples of proteindialyzed or tiLrat.ed back to 0.4 JI KCI, pII 7, according to themethod of Perry (40).

COOII-terminal anal\-


3/11

Subunit Structure of Myosin. III Vol. 244, Xo. 10I

10,590 rpmat low fringe displacements remains relatively constant. Dataobtained from experiments at 10,590 to 15,200 rpm indicate anaverage value of 468,000 (~10,000) for the molecular weight ofmyosin, at centrifugntion times from 44 to 71 hours (Table I).Somewhat higher values were obtained after 95 hours of centrifu-gation, prr*umably because of incomplete resolution of lown-mers.

Weight; average molecular weights were determined on thesame preparations of myosin by low speed sedimentation equilib-rium experiments. In one such experiment (Fig. 3), weightaverage molecular weights over the whole column were calculatedat intervals during centrifugation at 4,059 rpm. There is asteady fall iu values of I/M,,,, without significant change inthe second virial coefficient (Fig. 3, upper). Whc~l the zeroconcentration intercept, for M,,, is plotted against time ofcentrifugation (Fig. 3, lower), the weight average molecularweight. incareases linearly with time from 520,000 at the equilib-rium timr of 40 hours to 780,000 after 156 hours of ccntr ifuga-tion. (-4 precise extrapolation to zero time would require fur ther

I. . . . . I 1


4/11

Issue of May 25, 1969 L. C. Gershman, A. Strachm, and 1. Dreizeu 2729TABLE II

Apparent molecular weight of myosin in 0.4 M KCl-0.06 M KH#O,,pH 6.6, from approach to equilibrium and equilibrium

data obtained in scanner traces at 2800 AInitial hIolecular weight x 10-scdncentration Molecular weightx 10-b

5 min 20 min 2 hours 3 hoursw/ml I0.9 5.25 5.340.470.09

5.40 / 5.304.57 4.90 4.81 (hO.1)4.75 4.40 4.82 (f0.1)

5.40 4.w i 4.65 (f0.2)n Calculated by the method of Archibald, from regression lineof three-term power series on data from meniscus region, aftercentrifugation at 8,000 rpm for the indicated times.* From limiting slope of log concentration against r*, afterccntrifugation at 12,000 rpm for 48 hours, calculated as in Table I.

equilibrium experiments ou 30 successive preparations of unchro-mat.ographed myosin indicate that the light component disso-ciated at pl1 11 comprises 11.7% (~+~lw) of the protein (TableIII), and comparable results arc obtained with myosin chromato-graphed once on DEAE-cellulose or cellulose phosphate. Sedi-mentation equilibrium experiments indicate molecular weightsof 20,200 (&600) for the light subunits1 and 420,000 (~20,000)for the residual myosiu core (Table III). In a representativeexperiment, on cellulose phosphate-treated myosin at pH 11(Fig. 4), the molecular weight and the concentration of lightalkali component, are determined from the slope of log J againstr* at 39,330 rpm, and the molecular weight of heavy alkali com-ponent is calculated from the slope at. 13,330 rpm, after correctionfor the presence of light. component. The proportion of lightcomponent is bticd on determination of total protein from asynthetic boundary expcrimcnt.

Studies were also performed on myosin chromatographedTABLE III

hflect of chromatography on rabbit skeletul myosin

Ullchrornatographetf.l>EAE-cellulose, one cycle..Cellulose phosphateOne cycle... .. .._.Three cycles.

ATPau:activity -I50 0.4510 0.3 I

I Light alkali componentRatio, ( Scdimen-tationvelocity Sedimentation equilibrium, %::5 I :::i1.7 11.61.7

,1 9.0-

11.8 22,ooo8.0 j 25,200

-

-I-1

i

lkavy alkalicompment,sedimentationequilibrium-

mol Wf x 10-s4.2 + 0.2

4.3 * 0.24.4 + 0.3

information or assumptions on t,he kinetics of aggregation; an)such extrapolation would clearly be to a value somewhat lessthan 500,000.) In this experiment (Fig. 3), the rotor speed wasthen increased to 15,200 rpm, leading to meniscus depletionconditions in two of the three cells and, at equilibrium, the graphsof log J against rZ yield molecular weight values of 450,000 and465,000 (Table I). Other low speed sedimentation Lquilibriumexperimcuts indicated comparable findings on the time depend-ence of apparent molecular weight., confirmiug the occurrence ofcontinuous aggregation during cent,rifugation.

Experiments were also performed by the -Archibald method,in order to obtain molecular weight mc:lsurements early duringcent.rifugat.ion. The expcriment.s were performed at. proteinconcentrations less than 1 mg per ml with absorption opt.& anda photoelectric scanner at 2800 A, so as to minimize nonidealeffects that are appreciable at the higher protein concentrationsrequired for schlieren measurement,s by the Archibald method(6, 11). Successive determinat.ions of apparent molecularweight at the meniscus indicated values ranging from 535,000init,iallg to about 470,000 after several hours of centrifugat,ion(Table II). The change in M,,, is consistent with selectivedepletion of aggregated protein from the meniscus, sincbc nnjresidual effect from nonidculitg would cause an increase in M,,,as meniscus concentration decreased. At subsequent. cquilib-rium at 12,000 rpm t,he limiting slope of log concentration against,r* indicates a molecular weight of about 480,000 for myosin(Table II).

Dissoc-ialion of Light Subunits jrom Jlyosin Core--Data ob-tained from sedimentation velocit, and srdiment.ation

repeatedly on cellulose phosphate. The procedure is accom-panied by a slight decrease in specif ic hTPase, but no significantchange in the rat.io o f optical density at 2800 A to t.hat. at 2600 A(Table III). :1fter each passage through cellulose phosphateabout half of the chromatogmphed protein is recovered as solublemyosin; the remainder is found as a precipitate in 0.4 hl KC1 atpH 7, escept for a small amount presumably adsorbed on thecolumn. hfter three chromatographic cycles on cellulose phos-phate, about 10% of the original myosin remains soluble and thisfraction eshibits characteristic light and heavy alkali componentsof sedimentation velocity (Fig. 5~). IIowever, the proportionof light component is only 8 t.o 9% (Table III), and the averagesubunit weight is 25,300. Further evidence does not indicateany specific effect of cellulose phosphate in dissociating lightsubunits. Some of the chromatographed myosin that hasprecipitat,ed in 0.4 III KC1 at, pII 7 may be solubilized in 0.1 MK2C03-0.4 nr KC1 at, 1~11 11; the fraction shows light and heavyatkali components in customary proportion (Fig. %). Eurther-more, studies on the purified light subunits indicate their quanti-tative elution from the cellulose phosphate column under condi-tions identical with those used for the chromatography of myosin.Finally, light subunits may be dissociated from myosin at highsalt concentration at neutral pI1 (21, 37); the effect is highly

1 Use of the term subunit is based on the consistent, stoichio-metric presence of light component in preparations of nativemyosin, even after chromatography on DIME-cellulose or cellu-lose phosphate. This use is provisional, pending a precise de-termination of the functional role of light and heavy polypeptidechains.


5/11

2730 Subunit Structure of Myosin. III Vol. 244, No. 104 --

Ws? = 39,330 rpm2-%iti.c 1G LAC

5 CJ-= 4.22 (fO.2).- M =s 0.4 - 22,200L J,,: 0.50 (11.9%)Ei?g 0.2 - .0 I I I

8- WI = 13,330 rpm4-2-

2 O- -0.485 U = 9.32 (to.31E 0.2 - M = 22,200 M -428,000 _E J, = 0.50 (I 1.9%)so 0.1 I I I I I

m48(radial ~~+ancej2 50 2 51 bcm

FIG. 4. Fringe displacement, J, plotted against r2 from sedi-mentation equilibrium experiment on myosin (chromatographedonce on cellulose phosphate) at 1.08 mg per ml in 0.1 M Na2C03-0.4 M KCl, pH 11.0, at 4 in a double sector cell with duraluminum-filled epoxy centerpiece. UC, light alkali component; HAC,heavy alkali component; m, meniscus; b, bottom; M, molecularweight; JO, average concentration, in fringes. Upper, at 39,330rpm (~2). Lower, at 13,330 rpm (01). l , observed fringe dis-placement from meniscus position; 0, heavy alkali component(observed data less light component).specific, and phosphate salts are not among those causing subunitdissociation (Fig. 5~). The predominant effect of repeatedcellulose phosphate chromatography thus appears to be denatura-tion of myosin, and the procedure was not used routinely in thepurification of myosin.The light subunits are dissociated from myosin at pH valuesbetween pH 10 and pH 11 in 0.4 M KC1 (Fig. 7, upper). Aftertitration of myosin to pH 11.0 the light subunits are fully dis-sociated within the minimal time of 20 min that is requiredfor the formation of schlieren boundaries on sedimentation (Fig.5, f and g, and Table IV). At pH 10.5, however, the light sub-units are dissociated slowly (Fig. 54, and after 20 hours at pH10.5 the proportion of dissociated light component is only 6%of the entire protein.After further dialysis of alkali-treated myosin against 0.4 MKC1 at pH 7, sedimentation velocity experiments indicate reasso-ciation of light subunits with the myosin core (Fig. 5, e and h),the extent diminishing on prolonged alkaline treatment (TableIV). Comparable data are found on sedimentation equilibrium,with reassociation of 9% light subunits at pH 7 after short periodsat pH 11, and reassociation of only 2 to 4% light subunits at pH7 after storage for 3 to 20 hours at pH 11.

Sedimentation velocity experiments on myosin incubated at

FIG. 5. (upper). Sedimentation velocity of myosin, at 4 withphase plate angle 70. a, Soluble protein from third chroma-tographic cycle on cellulose phosphate, in 0.1 M NapCOa-0.4 MKCI, pH 11.0, at 52,640 rpm for 112 min, 6 mg per ml. b, Pre-cipitate from third chromatographic cycle on cellulose phosphate,dissolved in 0.1 M Na&O$-0.4 M KCI, pH 11.0, at 52,640 rpm for112 min: upper, 2 mg per ml; lower, 5 mg per ml. c, Myosin dia-lyzed against sodium phosphate, 0.5 M (upper) and 1.0 M (lower),at pH 8; at 52,640 rpm for 64 min, 8 mg per ml. d, Myosin storedin 0.1 M K&03-0.4 M KCl, pH 10.5, for 20, 3,0, and 1 hours (fromthe top down) ; at 50,740 rpm for 68 min, 5 mg per ml. e, Myosinst,ored in 0.1 M K&03-0.4 M KCl, pH 10.5, for 30 hours (upper)and 48 hours (lower), then titrated to pH 8; at 52,640 rpm for 96min, 6 mg per ml. f, Myosin stored in 0.1 M K&0$-0.4 M KCl,pH 11.5, for 20, 3, 0, and 1 hours (from the top down); at 47,660rpm for 48 min, 9 mg per ml. g, Same as f, at 128 min. h, Samesamples as inf and g, dialyzed against 0.4 M KCl-0.005 M NaHCOs,pH 7; at, 50,740 rpm for 56 min.FIG. 6. (lower). Sedimentation velocity on myosin, at 4 exceptfor b; phase plate angle, 70. a, Myosin in 0.4 M KCl, pH 6.5,stored at 40 for 24 hours (upper) and 1 hour (lower); proteinconcentration, 10 and 1 mg per ml in each cell; at 52,640 rpm for88 min. b, Myosin in 0.4 M KCl, pH 6.5, stored at 37 for 24 hours(upper) and 1 hour (lower) ; at 25 and at 48,000 rpm for 48 min, 9mg per ml. c, Same samples as in b, stored for 5 hours at 4 priorto experiment at 4; at 48,000 rpm for 104 min. d, Myosin storedin guanidine solutions, from top down : 2 M guanidine for 17 hours,2 M guanidine for 5 min, 1 M guanidine for 17 hours, 1 M guanidinefor 5 min; at 48,000 rpm for 228 min, 9 mg per ml. e, Second cycleprecipitate from 2 M guanidine-ammonium sulfate fractionation,in 5 M guanidine.IICl-0.4 M KCl-0.001 M EDTA; at 52,000 rpm for530 min: upper, 10 mg per ml; lower, 7 mg per ml. f, Third cycleprecipitate from 5 M guanidine-ammonium sulfate fractionationof carboxymethylated myosin, in 5 M guanidine.HCl-0.4 M KCl-0.001 M EDTA; at 52,000 rpm for 288 min: upper, 9.5 mg per ml;lower, 1 mg per ml. g, Third cycle precipitate from 2 M guanidine-&rate fractionation, in 5 M guanidine.HC1-0.4 M KCl-0.001 MEDTA; at 52,000 rpm for 408 min: upper, 9 mg per ml; lower ,4 mg per ml. h, Same sample as in g, dialyzed against 0.4 MKCl-0.05 M KHzPOh-0.001 M EDTA-0.001 M dithiothreitol, pH7; at 52,000 rpm for 48 min: upper, 8 mg per ml; lower, 4 mg per ml.


6/11

hue of May 25, 1969 L. C. Gershman, A. Strachu, and P. Ilreisen 273130-40 in 0.4 Y KC1 at, ~1-1 7 indicate dissociation into a lightcomponent (-3 S) and a predominant, heavy component (-8 S)(Fig. 6, a t.o c), with no significant reassociation ou return to 4(Pig. 6, b and c). In an esperiment at 4 on myosin earlierstored for 12 hours at 37, Archibald measurements at 8,000 to13,000 rpm indicate aggregation of the heavy component and,at sedimentation equilibrium at 13,000 rpm, its limiting molecu-lar weight is about 620,000. The data at 40,000 rpm indicateabout 6;; of a molecular w+ht 20,000 component and another4;/u of a molecular weight 90,000 component. Thus, heattreatment of myoairi would appear to result. in di?sociat ion oflight subunits from the intact. myosin core and irreversibleaggregation of the dissociated components.

Light. subunits ma?- also bc dissociated from myosin at guani-dine concentrat ions above.1 to 2 M (Pig. 64, and the full extentof dissociation is attained within the time required for the forma-t,ion of schlieren boundaries (Fig. 64. Sedimentation equilib-rium esperiments indicate the dissociation of light subunits, ofmolecular w-eight. 21,400 and c*omprising 12.0+Zc of the protein,from the intact myosin core, of molecular weight 430,000. Onredialysis against 0.4 LI KCl, 1111 7, sedimentation velocity andsediment&on equilibriunl experiments iudicate re:lssociat ion ofthe light. subunits with the myosin core, the extent, ranging fromtwo-thirds reassociation after I hour in 2 M guanidine to aboutone-half reassociation after 24 hours in 2 M guanidine.

EJect of Dissocinting Conditions on :I TPnse .4cti trity of Myoin-. On treatment of myosin above piI 10.0 follon-cd by titrationor dialysis to neutral pli, the ATPase activity of myosin is foundto be irreversibly lost, the extent of inactivation increasing onprolongtd alkaline trcatmcnt. As shown in Fig. 7 (upper), formyosin in 0.4 M KCl, ATlase in:&vation anti light subunitdissociation occur over comparable ranges in pII. At pI1 10.5ATPnse inactivation is faster than light subunit dissociation,

0.6 t\ 3 min 7 o- 121

2 M KCI-0.01 M ATP

10.0 10.5 II.0 II.5~ti

FIG. 7. pH dependence of light subunit dissociation andreversit)le ATPase activity. Myosin in 0.4 M KC1 (uppep) or 2 MKCI-0.01 M ATP (lower ) was titrated to a set pH with K&XI-KOIT solution. The percentage dissociation of light subunits(0) WRS determined from sedimentation velocity experimentsinitiated promptly after alkaline titration. ATPnsc activity(0) wa s determined on samples at alkaline pII for 3 min and 1hour, respectively, prior to dialysis against 0.4 M KCI-0.005 MNaHCOs, pH 7. Control ATPrtse activity was 0.55rmoIc of Pi permin per mg.

TABLE I\Time dependance of dissocialion and reassociation of

light ulknli componentSamples of myosin were stored in 0.1 M K&08-0.4 nr KCI, pII

11.0, for the times noted, and then dialyzed against 0.4 M KCI-0.005 M NaHCOa, pI1 7, for 24 hours.Light component

Time at pH 11

5 min1 hr3 hr20 hr

Sedimentationequilibriumb

At pH 11: After dialysis to pII i~_-SedimentationVelocitya Scd;nw&;ion- __- -

% %10.6 ( 4.812.1 7.512.0 8.710.6 8.7

%3 .oc8.010.0

a From schliercn patterns obtained within 20 to 60 min ofstat,ed times.* Experiments performed at 40,000 rpm; averngc molecularweight of light component is 25,000.c Data at 13,400 rpm indicnte :I molecular weight of 580,000for the heavy component.and at pII 11.0 :\TPase activity is totally lost within severalminutes. Inactivation of ATPase at pl t 11.0 is not preventedby EDTA, dithiot,hreitol, or 31~ ++-ATP, nor is activity restoredafter reduction with 0.2 M fl-mercaptoethanol. However, thereappears to be SOIW protection of ATI&c activity on alkalinetitration of myosin in 2 M KCl-0.01 JI .%lP (Fig. 7, Io~er), andapproximately 10% of coutrol ATIase may be recovered ondialysis to neutral pH after short periods at pII 11.0. The transi-tion zone for light subunit dissociation is broader in 2 M KCYl-0.0111 ATP than in 0.4 M KCl.

The protective effec t. of 2 nr KCl-0.01 M ATP n-as utilized toexplore the possible relationship between subunit compositionand :1Tlasc activity. Myosin at 6 to 8 mg per ml in 2 JI KCl-0.01 M AT ? wa s titrated to pH 11.0 with KgCOa-2 M KCI-0.01 MATP and salted out w ith 1 volume of 1.6 to 2.0 u potassiumcitrate, ~1.1 11.0 (26). Precipitate and supernatant were frac -tionated by centrifugation at 10,000 rpm for 10 min, and secondand third cnyc*le precipitates were obtaiiled by dissolving prccipi-tate in 0.1 M K&OI-2 M KCl-0.01 JI hTP, pH 11, and repeatingcitrate fractioilation. Identically treated samples of precipi-tates, supernntant, and unhactionated protrin were analyzed forATlase activity after simulta~leous dialysis against 0.4 M KCl-0.005 11 NnlIC03, pH 7, and for subunit composit,ion afte rdialysis against 0.4 M KCI-0.01 M Na?COa, pII 11 (Table V).The ATPasc activity is 0.1 pmole of Pi per min per mg, or 17%of control .YlPa+e, for t.he alkaline-citrate-treated but, unfrac-tionated myosin, whereas enzymic activity is markedly dimin-ished for the Ilart ially purified heavy chain core and is absent. inthe light subunits. Experiments were also conducted in whichsamples of citrate treated but unfractionntcd myosin were dilutedwith 1 volu~ne of freshly prepared supernntant (containing lightsubunit,s), 2 volumes of supernatant, or equivalent volumes of2 11 KCl-0.01 11 XTP-0.1 M K&Oa. On dialysis to ~1-1 7 thesolut.ions containing escc,ss light subunits wrre found to haveATlasc activity ap~~rosimatcl~ a-fold greater than the XTPascactivity of identically treated luyosill containing 124 lightcomponent.


7/11

2732 Subunit Structure of h+yosin. III Vol. 244, No. 10TABLE V

ATPase aclivily and subunit composition after alkaline-citrate ttealmenl and subunit fractionation

IractionResidual lightcomponent

ATPase activity@ _.-Stdimenta- Sedimenta-tion v&c- tion equilib-ity riumb

fin& Pi/mi?S/l?Sg0.100 (ZtO.017)

%12.0

7X11.7nfrnctionutedPrecipitate ~IIIS one-halfsupernatant.. . 0.071 (f0.020)Precipitate. 0.052 (f0.009) 5.5 7.2cSecond cycle precipitate 0.037 (f0.016) 4.4

Third cycle precipitate. 0.025 (f0.019) 3.9 G.4dSupernatant . 0 100~.0 Average (G.E.) based on five experiments; data are stand-ardized to 20 min of alkaline treatment prior to dialysis. ControlATPase is 0.G fimole of Pi per min per mg.b Experiments performed at 3G,OOO rpm; about two-thi rds of

t.he residual light subunits are present as a molecular weight20,ooO component and one-third as an aggregate of molecularweight -90,000. Sulfhydryl groups were not protected in theseexperiments.

c Data at 13,400 rpm indicate a molecular weight of 4.4 X lo6for myosin core.d Data at 14,000 rpm indicate a molecular weight of 4.5 X lO$for myosin core.TABLE VI

Kinetic data on heat denaturcllion of myosinI I Ik

ATPasse innctiva-tion..Light subunitdissociationa. .

SEC- SCG-~ 14.0 x 10-K 83 XW/1.1 x 10-s : 8.6 x 10-e

23 572.5 j 53

as:E.U.

10891

0 From schlieren patterns on sedimentation velocity at 4after heat treatment.Heat treatment of myosin also results in inactivation of

ATPase and light subunit dissociation (23), and the kinetics ofboth phenomena wa s studied during incubation of myosin at3040 over 24 hours. Both AIPase inactivat ion and light sub-unit dissociation follow first, order kinet,ics, with rate constantsfor ATPase inactivation greater than for subunit dissociation(Table VI). Values of activation enthalpy are about 55 kcalper mole for both processes, while the activation entropy isslightly greater for ;YlPase inactivation than for subunit disso-ciation (Table VI).

On treat.ment in 1 31 to 2 Y guanidine, myosin showed noATPase activity on furt .her dialysis against 0.4 Y KCl, pH 7;nor was ATPase preserved in the presence of EDT.%, dithio-threitol, or ATP.

Heavy Subunits of Myosin-The light subunits may be frac -tionated from the myosin core by alkaline dissociation followedby dilution in wate r al neut.ral pH (2); after three such cycles theproportion of light alkali component is reduced to 1 to 2% of the

protein (References 2 and 3 and Table VII). On dialysis ofthird cycle precipitate from reduced and carbosymethylatedmyosin against 5 M guanidine. HCl-0.4 M KCl, sedimentationequilibrium experimcnt,s indicate dissociation of t.he myosin coreinto heavy subunits of molecular weight 2 14,000 (Table VII).

In order to avoid possible elrects from alkaline hydrolysis ofpolypcptide chains, ultracentrifugal st.udies were also conductedon the myosin core fractionated iu guauidiuc solution. Aftertwo or three cycles of subunit. dissociation in 2 Y or 5 M gunnidineand salting out with ammonium sulfate or potassium citrate(see Methods), the residual precipitate fraction was dissolvedin aud dialyzed against; 5 M guanidine~HCl-0.4 M KU0.001 JIEDTA, pH 5.6. Sedimentation velocity experiments (Fig. 6,e to g) indicate I main component with BIL .s:,,,~ value of 4.4 8.In comparison with schliercn patterns of uufractionated myosinin 5 u guanidine (I, 3), there is less trailing behind the maincomponent, reflecting the removal of light subunits; an additionalleading edge arises from heavy chain aggregation (Fig. 6, f and8). The procedure which USILS 2 M guanidine and potaGumc&rule provided the best fractionation, and the proportion oflight subunits ~a.5 here reduced to 1 to 2c/b (Table VII). Sedi-mentation equilibrium data indicate liuear plots of log J against9, yielding a molecular weight of 211,000 for the heavy subunits(Table VII). Comparable vulues are obtained for mgosin audcarboxymethylated rnyosin.

Previous studies (41) on myosin have suggested that the heavysubunits are dissociated only at guunidine concentrationsapproaching 5 M, and this conclusion is confirmed in a sedimen-&ion equilibrium esperiment on third cycle precipitate in 4 Mguanidine, indicating a molecular weight of 414,000 for t.hepurified myosin core (Table VII).

Experiments were conducted on reconst.itution of the heavychain core after subunit dissociat.ion in 5 Y guanidine. Ondialysis of guanidine-treated third cycle precipitate against0.4 M KCl-0.05 N KH2P0,-0.001 Y dithiothreitol, pI1 7, sedimen-tation velocity experiment.s show a single self-sharpened bound-ary (Big. Gh), with extensive aggregation indicated by MI 11 Ssedimentation coeff icient and marked boundary asymmetry.Sedimentation equilibrium experiments indicate reassociation ofthe heavy subunits into soluble n-mers, w ith minimum molecularweight. on the order of l,lOO,OOO (Table VII). In similar experi-ments on the reconstitution of mgosin disociated in 5 Mguanidine but not fractionated into light and heavy subuuits,sedimentation velocit ,y experiments indicate a main boundarywith s j, w -8 S and about 3yo r&dual light component. Onsedimentation equilibrium (Table VII), the reconstituted myosinhas a minimum molecular weight, of about 580,000; the residuallight subunits undergo marked aggregation in the absence of0.001 .\I dithiothreitol.

COOH-k~minal ilnalyses-SSamples of myosin and partiallypurified heavy chain core obtained by alkaline fractionation showisoleucine ZE the predominant COOH-terminal amino acid andsubmolar quantities of several ot.hcr amino acids. In contrast,the purified heavy chain core obtained by means of five cycles of2 M guanidine-citrate fractionation shows only trace amounts ofseveral amino acids, even after digestion for 3 hours with carboxy-peptidase A.

DISCUSSIOSThe low speed sedimentation equilibrium data and the Archi-

bald measurements during early ccnt.rifugation indicate a weight


8/11

Issue of May 25, 1969 L. C. Gemhman, A. Stracher, and P. llreizenTABLE 1.11

2733

Sedimentation equilibrium experimenls on dissociation and reconstitution of heavy chain coreAll guanidinc solutions contain guanidinc.IICI-0.4 M KCI, pII 5.6; solutions used for guanidine-salt fractionation also contain 0.001 MEDTA.

Eractonaton procedure totor speed

In 4 M guanidine: third cycle precipitate, 2 M guanidine-ammo-nium sulfateIn 5 M guanidineThird cycle precipitate,* pi1 11, low ionic strength

Third cycle precipitate,6 pH 11, low ionic strengthThird cycle precipitate,b pH 11, low ionic strength

Average (Gi.D.)Second cycle precipitate, 2 JI guatlidirle-amnlonium sulfateThird cycle precipitate,c 5 M guanidine-ammonium sulfateThird cycle precipitatc,e 5 M guanidine-ammonium sulfateThird cycle precipitate, 2 JI grlanidine-citrate

Third cycle precipitate, 2 M guanidine-citrateAverage (G3.D.)In 5 M guanidine, then 0.4 ar KCI, pI1 7Third cycle precipitate,d 2 M guanidine-citrate

Third cycle precipitate,d 2 M guanidine-citrateMyosinhIyosin

-.- _rpm

36,99024,63019,16036,99021,72036,96020,38036,16020,12036,18022,02036,OCU18,00037,01021,74036,00024,00018,00037,02021,740

36,00013,0908,ooo14,2909,340

36,99013,41036,00014,000

-I Resdual light component I Heavy component0nwl rut %

18,700 3.518,709 3.518,706 3.520,06020,00021,00021,00019,50019,500

20,200 (zk700)19,30019,30018,60018,6002QP.50020,5002Q,mm,60020,60020,20020,20019, 100 (~1~800)

2.22.22.62.62.62.64.84.85.65.63.63.61.21.21.22.62.6

45,60045,60045,609

38,60038,60919,80019,800

0.40.40.4003.93.95.15.1

.J mol Wf x 10-r

(24.2)14.5 (4.17)4.14(28.7) (2.4)9.03 2.16(23.7) (2.0)7.78 2.11

7.59(22.5)

9.08

2.142.14 (~1~0.025)

(1.9)2.08

5.98 2.08S.68

(25.5)10.76.42W.6)8.98

2.07(2.2)2.102.21

(1.9)2.142.11 (rkO.054)

(27.0)9.04

(35.3)12.313.213.9

(12.8)11.3

(14.0)11.3

5.95.7

a Data obtained at high (T, indicated in parentheses, are not included in averages.b blposin reduced with 0.2 M B-mercantoethanol and carboxvmethylated.c Myosin carboxymethylatcd:d Solutions contain 0.05 u KHzPOd-0.001 H dithiothreitol.

average molecular weight of about 520,000 (G?O,OOO) fo r rabbitskeletal myosin, in accord with previous weight average valuesdetermined from light scattering (4, 5) and ultracentrifugal(6, 7) experiment.s on similar preparations of myosin. However,the evidence for the presence of aggregates in t,he original prep-arations of myosin (Table II) and t.heir formation during pro-longed centrifugation (Fig. 3) sugbests that the molecular weightof myosin is less than 520,000. Monomer may be resolved fromlow n-mers on high speed sedimentation equilibrium, and theresulting data indicate a molecular weight of 468,000 (~10,000)for myosin. That these experiments measure monomer n-eightis indicated by the invariance in results for at least 24 hoursbeyond equilibrium time and at rotor speeds over a range in Qfrom 6 to 13 (Table I). The 468,000 value is in close agreementwith the number average molecular weight of 470,COO (~10,000)reported by Tonomura, -%ppel, and Morales (i) from osmoticpressure measurements on myosin preparations not containingfree contnmiuants in significant proportion. -4 molecular weight

of 510,000 has also been determined on high speed sedimentat.ionequilibrium (9); the experiments were performed at a compara-tively low rotor speed (a - 4), however, and it seems possiblethat a small proportion of myosin dimers, prment originally orformed during 51 to 118 hours of centrifugation, may not havebeen fully resolved from monomer.

The value for s:,,~ of 6.1 S (Fig. 1) is at the lower end of therange from 6.0 to 6.43 S earlier reported for the sedimentationcoefficient of rabbit skeletal myosin (4, 8, 9, 42, 43). Althoughthe presence of a small proportion of low n-mers do= not appre-ciably affect the sharp schlieren peaks obtained on sedimentationof myosin, preliminary experiments by M. S. Drapkin, with theuse of scanner tracings at 2800 A, do indicate rapidly sedimentingmaterial in t.he plateau region; this component is proportionatelymuch smaller than a similar fraction noted on interferencepatterns o f sedimenting chicken breast myosin (44).

The over-all findings on the aggregation of myosin in 0.4 xKCl-0.05 M phosphate, pIT 6.5, are in accord wi th earlier obser-


9/11

2734 Subunit Stmcture of Jlyosin. II1 101. 244, Ko. 10pH 11

_ 2 M guanidine

I1M guanidine5 M guanidine -- ,z:

FIG. 8. Diagrammatic summary of subunit structure andinteractions of rabbit skeletal myosin. The stoichiomctric dat.aindicate 2.7 (M.3) light subunits per myosin molecule, with someevidence for selective association of 2.0 (f0.3) light subunits.Reassociation of light subunits is diminished on prolonged alkalineor guanidine treatment., an11 does not occur after heat dcnatura-tion.vations. In the most thoroughstudy onsl)olltnlleous:~ggregrationof myosin, Lowey and Holtzer (45) concluded that the phenome-non may involve a rate-limiting struct.ural change followed byrapid dimerization If higher aggregates may bc neglected, thisinterpretation yields :III equation (45) that siml)lifics to

J f, lf = 1 + kl + . . . , for U,/M < 2\vherc JI, is weight average molecuku weight,, .lI is monomerweight, and 1 is time. The low speed sedimentation equilibriumdata (Fig. 3) are in agreement with this model and iudicute a rateconstant, L, of 1.3 x 10e6 see-I, al 4. 111 ddition, the observedaggregation of cnrboxymcthylated myosin confirms the earlierconclusion (45) t.hat, intermolecular tlisulfidc bridges necul not beimplicated in the spontaneous aggregation of myosin. However,some kind of covsleut change appears likely, since aggregation isnot reversible at 4 sft,er prior storage at 30-40.

The preseut data indicate molecular weights of 420,000 for theheavy chain core and 212,000 for each heavy subunit. Previousfindings of homogeneity on ncrylamide gel elcctrophorcsis (46)and data on sulfhydryl pcptides (47, 48) would indicate that theheavy subunits of myosin WC similar if not identical, aud thisconclusion is supported by the invarinncc in hc:tvy chaiu weightat u values from 6 to 29 (Table VII). The absence of any evidentCOOH-terminal end group for the heavy chains suggests acyclical structure or an inaccessible amino acid. Alternatively,the occurrence of several end groups in submolar ratio wouldbe consistent with microhct~erogerleity of the heavy chains, andthis iutcrpretation appears to be favored in more recent ex-periments?

The light subunits arc 20,200 in average molecular weight,3P A. Stracher, unpuhlishcd data.a In a recent study on the purified light alkali componeiit ofmyosin, lrederiksen and IIoItzer (49) have reported molecularweight values o f 33,600 from light scattering mcasurcments in 0.3M KCI-0.1 JI phosphate, pH 8, and 31,600 from viscosity measurc-ments in 5 XI guanidine.HCl. The exneriments were done withoutprotection ocsulfhydryl groups, except for the use of 0.1 M &mercaptocthanol in some of the grianidine experiments. Theresults arc appreciably higher than the 20,000 value determinedfrom high speed sedimentation equilibrium data on light alkalicomponent (References 2 unt 3 and Table III) or acetylated lightcomponent (27). In evaluating the discrepancy in results , itshould be noted that the light component readilv undergoesaggregation and, in the absence of sulfhvdrvl protectfion, as muchas one-third of the light component may aggregate to the 60,000to 100,660 weight lcvcl (Tables IV and V). Aeereeation to thiscxtent.would result in weight average vr~lr of 2&06i to 45,000 fort.he light subunits. It is unlikely that heterogeneity of this kind

and comprise 11.7ci;, (&I 7;) of myosin prepared by the Szent-Gyorgyi method or chrolllatogral)hed once on DEAE-cellu1oseor cellulose phosphate. These data indicate 2.7 (~0.3) lightsubunits per myosin molecule (Fig. 8). In a somewhat lessprecise calculation, t.he diffcreuce iu molecular weight betweenmyosiu aud heavy chain core would indicate :I total weight ofapproximately 50,000 for the light COII~~OIIC~~, that is, about 2.5light subunits per myosiu molecule. The stoichiomctric datamust be iuterprcted with caution, however, since Locker andHagyard have reported quantitatively differeut electrophoreticbands for the light subuuits isolated from rabbit skeletal andcardiac myosin (50) and red uud white skclctal muscle (51),raising the possibility of Illicrohetcrogetlrit~ with two or threelight subunits per myosiu molecule.

There is also evideuce that two light subunits may be sclec-tivcly associated with the heavy subunits. The light, componenthas been earlier identified in heavy meromyosin (2, 27, 52), nudheavy meromyosiu IIMJ- be degraded into two particles of Sub-fragmeut 1 (32, 5355), each comprising remnants of oiic heav)subunit (mol wt 86,000) :md one light subunit (mol wt 18,000)in molar ratio (32). .Furthcrmore, after repeated cellulosephosphate chromatography (Table III) the residual solublemyosiu contains oiilg 8 lo 3; of light alkali component, or 2.0(~tO.3) light subuuits per myosiii molecule. If this finding maybe extrapolated to the bulk of dcnnturcd protein, it would appeart.hat, some of the light alkali component may be less tightly boundt.o t,hc heavy chain core or 111ny nut CWII be a11 essential part ofmyosin. In this regard myosin exhibits variable amouuts of5-adenylic acid deaminase (22, 26, 34, 44) and myokiuase(26, 44) activities; however, neither of these IWO proteins rcpre-sents a major fraction of the light alkali romponeut, for onpurific:ltion from rabbit skeletal muscle 5-adenylic dctlmiuasehas a scdimemat ion coefficient of 12 8 (56) :tnd myokinase hasdifferent sulfhydryl pept ides (18) aud COOH-termiual group(57) than the light, subunits of myosin. On the preyent evidenceit would thus appear that rabbit. skrletal myosin contains twolight subunits ( -9y0) with some certainty, and additional lowmolecular weight, material (2 to 37;) that may possibly reprrsenta less tightly bound light subunit, Irlicrohetcrogerleit~ of myosin,or trtcc coiitan~iiiaiits . .

With respect to the rcliort of Gaetjens et al. (26) that theproportion of light subunits is reduced to 4.8):, aftrr chromatog-raphy on IN%E-cellulose and cellulose phosphate, it should benoted that the 3.8y0 value reprericnts the recovrred yield of lightsubunits on their purificnt.ion (26), and is not. based on directult,raccutrifugal analysis of the entire myosin prcparatioii.Significantly, ullchrornntogrnl~hed myosin was found to yieldonly 3.2L; of light subunits (26), a proportion even less than thatdetermined for twice chrorn:ttogral,hed myosin. Also, thepublished schlicreu patterns (26) cm myosin aud twice chroma-tographed myosin show comparable proport ions of light compo-nent on succinylnt.ion. That t.he 4.8c/, vnhe may be attributedto incomplete recovery of light subunits rather than to chromn-togrnl)hic purification would be consistent with previous reportswould be discerned from the angular dependence of light scatter-ing measuremcnt,s, since the calculated dissymmetry is only 1.01for a sphere of 166.4 diameter or a cylinder of 156 A length (verycrudely approximating an n-mer of molecular weight 100,000).In any case, light scattering and viscosity measurements on anaggregated syst.em are clearly referable to an average value, andone may qiiestion whether the higher molecular weight vtl~ms (40)describe light chains that, arc in part aggregated.


10/11

Issue of May 25 > 19W I,. C. Gershnzan, A. Stracher, and I. Dreizen 2735(2, 3, 14, 15) that the yield of light. subunits isolated by solubilitymethods is apl)reciably 1~5s than the proportion detcrmincd onultracrntrifugal analysis.

The proposed model for native myosin (Fig. 8) indicates aweight average molecular weight of 18i,OOO IO 190,000 on com-plete dissociation into light and heavy polypeptide chains.This value may be compared with the weight average molecularweight of 19i,C@O that, was determined by Woods, Himmclfarb,and IIarritlgtoll (1 I) from low speed sedimentation equilibriumexperiments on mywin in 5 JI guanidinc. HCl. These esperi-mcnts (11) were contlucled on myosin that ~;II; unfractionatcdwith respect to light wbuuits and ~)rcsum:tbly contained mostif not all of the light component of native myosin. In supportof this interpretation, it. has been noted earlier (3, 12) t.hat:\rchihald experiment.s (I 0) and sedimentation velocity esperi-merits (46) on identical lxeparations of myosin are suggestive oflow molecular weight hetcrogelwity in gunnidiue and ureasolutions.

The dissociat. ioll of light subunits at l)I-I I1 and in 2 JI guanidineawonipanies r:il)itlly owurring struct.ur:il changes in myosin,manifest in bot,h (*a~: by inrreaae in levorotat ion and decreaseill intrinsic viscosi ty (-II, 58). These changes presumablyinvolve an unfolding of the helicul conforni:ltion in niyosiii (-II),although some of the fall in intrinsic viscosity is attributable tosubunit dissori:it.ioii. I )wl)i te nearly comp1t:t.s unfolding in 3to 4 M guanidino (1 I), the heavy chain core remains intact atguatiitliile coiiwntratiolls up t.0 4 M. .-\ ftcr light subunit di>so-ciatioii at: 111 11 or in 2 11 guanidine, at least two-thirds of thelight subunit:: reawwia tc with the he:lvy chain core on subs~qiient dialysis ag:lin+t 0.4 XI KCI, l)t 1 7 (Fig. 8). Completerm?;sociat ion al)pwrs to be prevent cd by changer, possiblycovalent Fide chaitl re:tct.iow, that occur OJ~ prolotlged alkalineor gwnidinc trcat.nncnt, a11t1 in the c:we of heat dennturationthere is no reaxsoc:iat.ion at all. Light subunits tend to aggregateill the absenw of sulfhydr>.l lxotection; howvcr , subunit rcasso-ciation is not. augnwnlcrl in the lnwtwcc of 0.001. M dithiothrcitolor after prior carhosyinct hylation of niyosin.

The present data confirm the report (-II) that. the heavy chaincore tnay br rwonst ituteti after dissociation in 5 M guanidine(Fig. i), :11x1 thrrc is some ovitlctw that aggregation to lown-incrs is cwtiallccd in the abs;cnc,c of light wbunil~. Whileguauidinc-i rratccl rn\oGn 111x?- be rcconst itutcd with at leastsome of the protein at, the 500,000 to 600,000 weight. lcvcl (Refer-enw 41 and Table VI I), the guaJlidilic~-tlc~ltctl but fractionatedheavy subunits arc rcronstitutetl with ;I mil~irnuni weight on theorder of I ,100,oW (lal)lr TII). The purifietl heavy chain coreaggrcg:itcs to :I >imil:ir lcvc~l after the rcnwv>il of light subunitsby alkaline fr:ic:tioi~al ion (2, 3). Purttirrniorc, rrconst itutioncslwiment~ on g~J:lllitliJlr-trc,Rted nlyosin have indicated extcn-sivc aggregnt ioll inwIving intcrmolrcwlar tlisulfidc bridges (41);howwr, the I~e:lvy chain core undrrgocs :~ggrcgation in 0.4 MKCI at pH 7 after lwior retluct ion and c~:lrt)osrJleth:lutioJl (2,3), in 0.001 M dithiothrcilol (Table VII), :IJI~ without sulfhydryll)rotwt.ion (T:ibIe VII). The fintlings >u,,lw-;t. that removal oflight subunit> rna\~ uncover -ites on the hexvy c+~:lins that enterinto aggrcgat iota I)hcnomcnn, prcwn1:thl\- vi:1 disulfide hridgce,othrr covalent bondr, or both.

The location of light. subunits in hrxvy mrwmyosin (2, 27, 52)a~rtl Subfr:~gnxnt 1 (32) raises the possibility th:lt the light sub-iiliits arc involved in the biological activity of nlyosin. Thedissociation of light. subunits from the l1wv.v chain core at plI

11; in 2 31 guanidiue, a11d 011 heat treatment is accompanied bythe inactivation of .\TPase, indicating that both .\TPase inac-tivation and light subunit dissociation may follow commondcnnt wing procws rs. The values of AHI and AS: for bothphenomena are comparable with values reported 011 heat dena-trwtion of other proteins (59), and the more rapid rate forAllasc inactivation is maniftst by a slightly grcatrr activationentropy for subunit dissociation. That subunit interactions andhLPase activity may be more essentially related is furthersuggested by the esperiments involving alkaline dcnaturation ofIl1yosin in 2 ar KCl-0.01 11 .YIl (Table T). There appear-r; to ber;ome recovery of IYT1:~.se act ivit,y in rcconst it ut cd my&n,which is lwt on removal of light subunits and augmented onaddition of freshly dissociated light subunits. The resultssuggest, that the ATPax activity of myosin may involve somekind of interaction between light and heavy subunits, and thequestion will be further esl~lorcd in a subsequent comniunical.ionOJI the lwolwties of myosin in concentrated salt solutions.

lGn:~lly, we may conxirler whether the light ant1 heavy poly-lwl)tidc chains of JIU~ ivc myosin belong to a single molecule orwhether native mywin comprises several clowly associated butdistinct ~noleculcs. The present el-idencr would appew to favora structure of two axially symmetric, eiizymiwlly active pro-tomers, each comprising otle heavy and one light subunit, with,211 ultlit~ion:d 2 to 35:; of light. component that, wmains undefined.This in1 rrpretation derive5 from the presence of light and heavysubunits in molar ratio in each particle of Subfragment. 1 (32),the Ftoichiometric presence of two light subunits aft or wpeutedcctllulor;~: pho~l)hate chromatogral)hy of mywin (Table III), theclwe relationship belwccn light subunit dirsnriat ion andirrc~waible .iTPasc in:wti\-xt ion (Refrrcnw 40, Fig. 7, andTable VI), the dependenw of partially rrgcneratctl .Yllaseactivity on subunit composition after alkaline trcatmcnt in 2 MKCI-0.01 M ATP (Table V), and the marked agjircgation of thepurified heavy chain core in 0.4 M KU, pH i. Scverthclcss, itwould be premat.ure to reach a final conclusion on this evidencealone, :111d he above hypothesis is under active inw


11/11

2736 Subunit Structure of Myosin. III Vol. 244, So. 105. ITOLTZEX, A., LO~EY, S., ASD SCHUSTER, T . XI., ilfokcularbasis of neoplasia, University of Texas Press, Austin, 1962,

p. 259.G. %~UELLER, H., J. Viol. Chem., 238, 797 (1964).7. TOSOMURA, Y., APPEL,P.,.\ND~~OHAI,ES, M.F.,Hiochemislry,6, 515 (1966).8. TIUYEH, I. P., AND PERRY, S. V., Biochcm. Z., 346,87 (1966).9. CHUXG, C.-S., RICIURDS, E. G., .~ND OLCOTI., II. S., Rio-chemistry, 6,3154 (1967).10. KIELLEY, W. W., ,\ND I~ARRIN(;TON, W. E., Z?ioch im. Biophys.Acta, 41, 401 (i960). 38.11. WOODS. E. F.. HIMMEI.F.4RB. S.. ASD ~~ARRISCTOX. W. P..J. Biol. Chek., 238, 2374 (lci63):12. STRXIIRR, A., .~NI) DREIZEN, P., Curr. Top. Z?ioenerg., 1, 153(1966).13. Tsno, T. C., Biochim. Biophys. Acla, 11,368 (19.53).14. KOMINZ, D. R., CI\RROI,L, W. R.,SMITH,II., IIIRRINGTON, W. F., .\ND KIELLEY, W. W.,J.Hiol.Chem.,237, 3116 (1962).JOHNSON, I'., ;\ND ROWE, A. J., Hiochem. J., 74. 432 (1960).biOUM.IERTS, W. F. H. &I., ASD ALDRICH, B. B., Biochim.Biophys. Acla, 28, 627 (1958). RICHAI~DS. E. Cr.. CHUNG.C. S.. MENZEI.. 11. B.. ANI) OIXOTT.II. S., Lkochekistry, 6; 528 (i967). LOWEY, S., AZW HOI,~IZEH, A., J. Amer. Chew Sot., 81, 13%(1959).SMALL, P. A., HARRINCTON, W. E., ASI) KIE:LLE:Y, W. W.,Z3iochim. Biiphys. Acla, 49; 462 (1961).KIELLEY. W. W.. ASD BARNET~'. I, . M.. lliochim . BioDhvs. Acla.

Documents

J. Biol. Chem.-1969-Gershman-2726-36