Subunit

III.

Structure
RIODEI,

of Mvosin
J

A PROPOSID

FOR

RABBIT

SIII<T,ET,1T,

>IIEOSIN*
(Received for publication, November 12, 19G8)

LEWIS
Prom

C. GERSHMAN,:
the Departments

X.

STI~ACHEH,#

ASI)

I.

DHEIZES~

Brooklyn,

of Medicine New York 11203

and Biochemistry,

State Unirerhg

of Xew Yodi Downstate Uedical

Center,

SUMMARY High speed sedimentation equilibrium experiments indicate 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 in ultracentrifugal experiments on the same preparations of myosin. At pH 11, in 2 M guanidine, and on heat treatment, myosin is dissociated into a core of molecular weight 420,000 weight and light subunits of average molecular weight 20,200. The core may be dissociated by 5 M guanidine into two heavy subunits, of molecular weight 212,000 (=tS,OOO), having no evident COOH-terminal group on digestion with carboxypeptidase A. The light alkali component comprises 11.7% ( f 1%) of the protein, or 2.7 ( hO.3) light subunits per myosin molecule, for unchromatographed myosin and myosin chromatographed once on diethylaminoethyl cellulose or cellulose phosphate; the molar ratio is reduced to 2.0 (hO.3) in a small fraction of the original myosin that is recovered after three chromatographic cycles on cellulose phosphate. Myosin may be reconstituted after subunit dissociation at alkaline pH or in guanidine solution; however, the reassociation of light subunits is diminished after prolonged denaturation. The removal of light subunits is accompanied by aggregation of the heavy chain core to low n-mers. In general, those denaturing conditions which lead to light subunit dissociation also result in the irreversible inactivation of adenosine triphosphatase and, on heat denaturation, values of AHf and AS: are comparable for both processes. The ATPase activity of myosin may be partially protected on alkaline treatment in 2 M KCl-0.01 M ATP; enzymic activity is lost in identically treated but
* This stlidy was supported hv Rcscarch Grants U-13(X (to P.I>.) from th& Ilcalth it&arch C!ouncil of the City of ?;ew Ydrk and GM-07076 (lo A.S.) and ARI-001G.5 (to P.11.) from the United States Public Health Service. This work was taken in part from a thesis submitted by L.C.(;. for the dcgrcc of Doctor of Philosophy in Phvsioloev and Bioohvsics. State Univcrsitv of Kcw York. Porlidns of zis work w&e pre.&nted at the 13thAnnual Meeting of the Biophysical Society (W, Gl). The preceding paper in this series is Reference 2. $ hIcdic~ul Scientist Fellow of the Lift Tnsurrtnce Medical Research Fund. 0 Career Scientist of the IIenlth Research Council of the City of Se\\- York.

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

in the

Previous st.udies (I-3) i11 t.his series indicate that at. pII II about three light or globular subunits of molecular weight 20,000 may be dissocint.ed from the heavy meromyosin end of rabbit skeletal myosin, and i.he r&dual protein may bc dissociated b3 5 nr guanidine into two heavy or fibrous subunits of molecular weight 210,000 to 220,000. .\Iost recent experiments indicate a molecular weight of approximately 500,000 for rabbit skeletal myosin (49). While appreciably higher xxlues (10, 11) ma> result from aggregation of myosiii (3, 5, 12), some uncertainty itpelf reflect minor remains nhrt.her the 500,000 ~11ue 111:q degrees of aggregation and, in this respect., wight average molecular weights of 480,000 to 524,000 (4-7) do exceed ii single numbrr average value of 470,000 (i). Sirwe knowledge of monomer weight is important in the interpretation of subunit, comlwsit ion, the quest.ion has been explored by means of high speed and low speed wdimentation equilibrium esperiments on identical preparations of myosin; prcliminay data have bern reported (2, 3). A low molecular wcight component may be di*wriatcd from myosin at alknlinc pH (2, 3, 13-IS), in 5 Y gunnidine (l-3, 13), in urea solutions (3, 13, 19, 20), in concentrated ealt solutions at neutral 111 (21), and 011 heat crcat.ment (16,22, 23), succinylntion (24-26), and acetylation (16, 27). The light component comprises about 127; of unchrorllntogruphed rnyo&l (2, 3), has a well defiurd amino acid comlmsition (14, 19, 26, 27), and has COOH-terminal isolrucine (2, 14, 18, 22). The rondit,ions leading to the diswciation of light component from native myor;in also result. in the inactivation of adcnosine triphosl)hatase, and attempts have been made at chromatographic separation of t.he light component from myosin preparations. Although the light component is still precut. after chromatography on diethylaminoelhyl cellulose (27, 28) and DEAE-Scphades (17, IS), it has been reported (26) t.hat myosiu contains only 4.576 of lo\v molecular 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

Issue

of May

25, 1969

L. C. Gershman, A. Stracher, and P. Dreizen

2727

components of unc:hromntographed and chromatographed myosiii. Studies were also conducted on the time de1xndence of dissociation and reassoci:Aon of light component with the heavy chain core, aud on possible protection of ATPase during such 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 been on preparations containing, or very likely containing, light component in molar 1)roportion or greater. Ultracentrifugal espcriment.s and COOI-I-terminal analyses on the purified heavy subunits arc here dcacribcd. METIIODS Rabbit. skeletal myosin, prepared essentially by the method of Szent-Gybrgyi (1, 33), was stored in 0.4 M KC1 at pI1 6.5 until use. Some prc1)nrations were further chromat.ographed on DE.U!-ccllulo~c (34) or celluloac phosphate (Signla) (26, 20) by detailed proccclurci; most experiments were done 011 uiichromatUgraph(,d myosin. The procedures used for ~1udies on myosin aL equilibrium in 5 Y gu:midine and at pI1 11 have been described (1, 2). In experiments on the time tlcpendence of alkaline denaturation, the protein solution was t.itrated to alkaline pH with li,CO1KOII solution, and reversibility was studied after back tit.rat.ion or dixlgsis to neutral pFI. For comparable espcrimcnts in 1 M or 2 JI guanidine, concentrated guunidine-0.4 JI KC1 was added to prot,ein solut.ioll, and rever~ibi1it.y was studied after dialysis against 0.4 M KCI-0.005 nr NalICOs, pII 7. IIeattreated mywin was incubated in a water bath at 25-40 for periods up to 24 hours. The heavy subunits of myosin were purified by one of the following methods: (a) alkaline fractionation (2), in which protein 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 dilution with water, and (b) guanidine fractionation, in which the protein was treat.etl in 2 M or 5 M gunnidine.HCl-0.001 M EWJh for periods up to 24 hours and xdtcd out with ammonium sulfate (35 to 45% fract,ion) or 0.8 M potassium citrate (26), and the precipitate was wwhcd in dist.illed water. Two or three cycles were performed for each 1)rocedure. T~ltrnccnt.rifugal esperimcnts were done at, 4 in a Beckman model E analytical ultraccntrifugr equipped with xchlirren and interference optics and, for the later work, with electronic speed control. III sedimentat.iou velocity experiments the prol)ortions of light and heavy components were det.ermined from schlieren measurements corrected for radial dilution and JohnstowOgston effect (2, 35). IIigh speed sedimentation equilibrium esperimerits wwc done by the method of Yphnntis (36); multicomponent analysis is based on data obtained at successive equilibria at two or more rotor speeds, as clwribcd elsewhere (2, 37). In the experiments at pH 11, after heat treatment, and in 2 31 guanidinc, equilibrium data were customarily obtained after 29 to 40 hours at 13,000 t.o 14,000 rpm and after 14 to 24 hours at 36,000 Lo 40,000 rpm. Corrections for Wiener skewing (36) were negligible. I,:nv speed sedimentation equilibrium cy)eriments were analyzed by the whole column method (38), and .\rchibnld measurements are based on combined schlicrewinterfercnce measurwwnts (39). Absorption optics and a photoelect,ric w:tmwr were II& in several experiments that are dexribed

5 concentration

IO mg/ml

15

20

PIG. 1. Reciprocal of sedimentation coeficieut (SW-~) plotted against initial concentration of myosin, in 0.4 M KCI-0.05 11 K&PO,, pH G.5, at 4. esplicitly. Equilibrium experiments were usually done with 12-mm double sector durnluminum-filled or chorcoa-filled Epon centerpieces; a sis-cornpnrt.lneiIt charcoal-filled Epnn centerpiece was used in some of the low speed equilibrium espcriments. Synthetic boundary experiments were done wit-h twin capillary cent.erpieces, at. protein conccnt~rations of 3 to 6 mg per ml. Protein conccntrat.ion was dctcrmincd from optical density at 2800 A measured with a Zeiss NIQ II spectrophotometer md corrected for Rnyleigh scattering. Value for extinction coeff~cicnts, refractive index increments, and partial specific volume of myosin were as noted (1, 2). Values were assumed to be ident.iwl for the purified heavy subunibs. The extinction coeffXcnt 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 protein dialyzed or tiLrat.ed back to 0.4 JI KCI, pII 7, according to the method of Perry (40). COOII-terminal anal\-<es were done on samples of protein digested with cnrbosypeptidasc A, as previously described (31). RICSULTS 3lolecukzr Weight of Xyos%-In 0.4 M KCl-0.05 JI KlI$O,, pH 6.5, myosin sho\vs a single sharp peak on sedimentation velocity. The sedimentation coeficieut is inversely proportional to protein concentration (Fig. l), fitting the equation
G.13S s20

IW = 1 + o.G99c

where c is concentration, in milligrams per ml. The boundary occasionally shows a small leading edge, notably after storage of myosin for longer than several days; however, discrete leading or trailing peaks are not found in preparations obtained by the Szcnt-Gyiirgyi method or furt.her chrnmatngraphed on DEAEcellulose or cellulose phor;phate. In a representative high spcxtl scdiment.ation equilibrium espcriment on mynsin (Fig. 2), in which the. expected equilibrium time (37) is on the order of 40 hours, the slope of log J against r2 is linear at 0.2 to 2 fringes; the slope is nonlinear at higher fringe displacements, reflecting the presence of soluble aggregates of myoPin. On prolonged cent.rifugat.ion, the conrent ration distribution shifts t.ow:ud the bottom of the cell and a marked incre:ise in slope at. high fringe values indicates continuing aggrcg,at ion of mynsin. .\ggregation is sufficiently slow that the slope

Subunit
I

Structure

of Myosin.

III

Vol. 244, Xo. 10

10,590

rpm

at low fringe displacements remains relatively constant. Data obtained from experiments at 10,590 to 15,200 rpm indicate an average value of 468,000 (~10,000) for the molecular weight of myosin, at centrifugntion times from 44 to 71 hours (Table I). Somewhat higher values were obtained after 95 hours of centrifugation, prr*umably because of incomplete resolution of low n-mers. Weight; average molecular weights were determined on the same preparations of myosin by low speed sedimentation equilibrium experiments. In one such experiment (Fig. 3), weight average molecular weights over the whole column were calculated at intervals during centrifugation at 4,059 rpm. There is a without significant change in steady fall iu values of I/M,,,, the second virial coefficient (Fig. 3, upper). Whc~l the zero concentration intercept, for M,,, is plotted against time of centrifugation (Fig. 3, lower), the weight average molecular weight. incareases linearly with time from 520,000 at the equilibrium timr of 40 hours to 780,000 after 156 hours of ccntrifugation. (-4 precise extrapolation to zero time would require further . 51.2 cm2 . 51.4
a

I. 50.6

. . 50.6 51.0 (radial distanceI

I
2 0

<

4.059

rpm

, I- 24 T ,- 36

FIG. 2. Fringe displacement, .I, plotted against sqllare of radial displacement, ~2, from sedimentation equilibrium experiment on mynsin at 0.96 mg per ml in 0.4 M KCl-0.05 M KH~IOI, pH 6.5, at 4 in a double sector cell with charcoal-filled Epon centerpiece. Meniscus was at 47.905 cm2; bottom at 51.610 cm*. Calculations were based on photographs obtained after 44, 50, 67, 81, and 94 hours of centrifugation at 10,590 rpm.

f 0

TABLE
High

I
on

speed sedimentation equilibrium experiments myosin in 0.4 M KU-O.06 M KHIPO,, pH 6.6

4 concentration

6 mg/ml

The molecular weight values are based on the analysis of 10 to 20 data points, each representing an average of three to five independent comparator readings. Weight average molecular weight was determined from the linear slope of log J against r* at low concentrations. Equivalent results were obtained by a least squares analysis in which J values were weighted according to their variance. All calculations were done with an Olivetti-Underwood 101 computer.

Rotor speed

I
45 f 2

MO ocular weight X 10-S determined centrifugation for hour

7 f 2

by

-*pm

hour ._-.

10,590

11,x3@ 14,390 15,200

4.64 4.57 4.76 4.40 4.90 4.76 4.61 4.56

4.70 4.57 4.74 4.40 4.87 4.79 4.88 4.73 4.71 f 0.056

4.70 4.81 4.86 4.55 4.71 4.65 4.50 4.68 f 0.049

5.1 4.91 5.1 4.59

50
centrifugotion

too
time, hours

150

Average

S.E.

, 4.66 f 0.060

4.92 f 0.120 --

0 Reduced

and carboxymethylated

myosin.

FIG. 3. Sedimentation cynilibrium experiment on myosin at 1.7, 2.9, and 6.5 mg per ml in 0.4 of KCl-0.05 M KHIPO,, pTI 6.5, at 4 in a double sector cell with a six compartment charcoal-filled Epon centerpiece. The aqueous solutions, of column height approximately 1.8 cm*, were layered over FC 43 oil, column height approximately 0.8 cm*. Apparent molecular weight (&I,,,,) was calculated over the whole column from photographs obtained at 24 to 156 hours of centrifngation. Expected equilibrium time (37) is about 40 hours. [Jpper, reciprocal of apparent molecular weight plotted against initial protein concentration; lower, weight average molcc!nlar weight (M,), from extrapolation to zero concentration in upper graph, plotted with respect to ccntrifngation time.

Issue

of May

25, 1969
TABLE II

L. C. Gershman, A. Strachm, and 1. Dreizeu

2729

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 A
Initial cdncentration 5 min w/ml I hIolecular 20 min weight x 10-s 3 hours Molecular weight x 10-b

2 hours

0.9
0.47 0.09

5.25 5.40 5.40

! 5.34 / 5.30 4.w

4.57 4.75

4.90 4.40 i

4.81 4.82 4.65

(hO.1) (f0.1) (f0.2)

n Calculated by the method of Archibald, from regression line of three-term power series on data from meniscus region, after centrifugation at 8,000 rpm for the indicated times. * From limiting slope of log concentration against r*, after ccntrifugation at 12,000 rpm for 48 hours, calculated as in Table I. TABLE
hflect of chromatography

equilibrium experiments ou 30 successive preparations of unchromat.ographed myosin indicate that the light component dissociated at pl1 11 comprises 11.7% (~+~lw) of the protein (Table III), and comparable results arc obtained with myosin chromatographed once on DEAE-cellulose or cellulose phosphate. Sedimentation equilibrium experiments indicate molecular weights of 20,200 (&600) for the light subunits1 and 420,000 (~20,000) for the residual myosiu core (Table III). In a representative experiment, on cellulose phosphate-treated myosin at pH 11 (Fig. 4), the molecular weight and the concentration of light alkali component, are determined from the slope of log J against r* at 39,330 rpm, and the molecular weight of heavy alkali component is calculated from the slope at. 13,330 rpm, after correction for the presence of light. component. The proportion of light component is bticd on determination of total protein from a synthetic boundary expcrimcnt. Studies were also performed on myosin chromatographed

on rabbit

ATPau: activity

III
skeletul myosin
Light Scdimentation velocity alkali component

Ratio,

I
(

Sedimentation

equilibrium

I -1

lkavy alkali compment, sedimentation equilibrium

Ullchrornatographetf. l>EAE-cellulose, one cycle.. Cellulose phosphate One cycle..... Three cycles.

, % ::5 I :::i
.._. 50
10 0.45 0.3 -I 1.7 1.7 , 1 11.6 9.0
-

mol Wf x 10-s

4.2 + 0.2

11.8 8.0

22,ooo 25,200

4.3 * 0.2 4.4 + 0.3

information or assumptions on t,he kinetics of aggregation; an) such extrapolation would clearly be to a value somewhat less than 500,000.) In this experiment (Fig. 3), the rotor speed was then increased to 15,200 rpm, leading to meniscus depletion conditions in two of the three cells and, at equilibrium, the graphs of log J against rZ yield molecular weight values of 450,000 and 465,000 (Table I). Other low speed sedimentation Lquilibrium experimcuts indicated comparable findings on the time dependence of apparent molecular weight., confirmiug the occurrence of continuous aggregation during cent,rifugation. Experiments were also performed by the -Archibald method, in order to obtain molecular weight mc:lsurements early during cent.rifugat.ion. The expcriment.s were performed at. protein concentrations less than 1 mg per ml with absorption opt.& and a photoelectric scanner at 2800 A, so as to minimize nonideal effects that are appreciable at the higher protein concentrations required for schlieren measurement,s by the Archibald method (6, 11). Successive determinat.ions of apparent molecular weight at the meniscus indicated values ranging from 535,000 init,iallg to about 470,000 after several hours of centrifugat,ion (Table II). The change in M,,, is consistent with selective depletion of aggregated protein from the meniscus, sincbc nnj residual effect from nonidculitg would cause an increase in M,,, as meniscus concentration decreased. At subsequent. cquilibrium 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 obtained from sedimentation velocit,! and srdiment.ation

repeatedly on cellulose phosphate. The procedure is accompanied by a slight decrease in specific hTPase, but no significant change in the rat.io of optical density at 2800 A to t.hat. at 2600 A (Table III). :1fter each passage through cellulose phosphate about half of the chromatogmphed protein is recovered as soluble myosin; the remainder is found as a precipitate in 0.4 hl KC1 at pH 7, escept for a small amount presumably adsorbed on the column. hfter three chromatographic cycles on cellulose phosphate, about 10% of the original myosin remains soluble and this fraction eshibits characteristic light and heavy alkali components of sedimentation velocity (Fig. 5~). IIowever, the proportion of light component is only 8 t.o 9% (Table III), and the average subunit weight is 25,300. Further evidence does not indicate any specific effect of cellulose phosphate in dissociating light subunits. Some of the chromatographed myosin that has precipitat,ed in 0.4 III KC1 at, pII 7 may be solubilized in 0.1 M K2C03-0.4 nr KC1 at, 1~11 11; the fraction shows light and heavy atkali components in customary proportion (Fig. %). Eurthermore, studies on the purified light subunits indicate their quantitative elution from the cellulose phosphate column under conditions identical with those used for the chromatography of myosin. Finally, light subunits may be dissociated from myosin at high salt concentration at neutral pI1 (21, 37); the effect is highly 1 Use of the term subunit is based on the consistent, stoichiometric presence of light component in preparations of native
myosin, even after chromatography on DIME-cellulose or cellulose phosphate. This use is provisional, pending a precise determination of the functional role of light and heavy polypeptide chains.

2730
4 -Ws? = 39,330 2% iti .c 1 G 5 .- 0.4 s L
E i? g 0.2 I 0 I WI = 13,330 rpm rpm

Subunit

Structure of Myosin.

III

Vol. 244, No. 10

LAC

CJ-= 4.22 (fO.2) M = 22,200

J,,: 0.50 (11.9%) . I

842-

E 0.2 E
s o 0.1 I m48

O- -0.485 M = 22,200
J, = 0.50 (I 1.9%) I I I

U = 9.32 (to.31 M -428,000 _

I

50
(radial ~~+ancej2 cm

51

FIG. 4. Fringe displacement, J, plotted against r2 from sedimentation equilibrium experiment on myosin (chromatographed once on cellulose phosphate) at 1.08 mg per ml in 0.1 M Na2C030.4 M KCl, pH 11.0, at 4 in a double sector cell with duraluminumfilled epoxy centerpiece. UC, light alkali component; HAC, heavy alkali component; m, meniscus; b, bottom; M, molecular weight; JO, average concentration, in fringes. Upper, at 39,330 rpm (~2). Lower, at 13,330 rpm (01). l , observed fringe displacement from meniscus position; 0, heavy alkali component (observed data less light component).

specific, and phosphate salts are not among those causing subunit dissociation (Fig. 5~). The predominant effect of repeated cellulose phosphate chromatography thus appears to be denaturation of myosin, and the procedure was not used routinely in the purification of myosin. The light subunits are dissociated from myosin at pH values
between pH 10 and pH 11 in 0.4 M KC1 (Fig. 7, upper). After titration of myosin to pH 11.0 the light subunits are fully dissociated within the minimal time of 20 min that is required for the formation of schlieren boundaries on sedimentation (Fig.

5, f and g, and Table IV). At pH 10.5, however, the light subunits are dissociated slowly (Fig. 54, and after 20 hours at pH 10.5 the proportion of dissociated light component is only 6% of the entire protein. After further dialysis of alkali-treated myosin against 0.4 M KC1 at pH 7, sedimentation velocity experiments indicate reassociation of light subunits with the myosin core (Fig. 5, e and h), the extent diminishing on prolonged alkaline treatment (Table

IV).

Comparable data are found on sedimentation equilibrium,

with reassociation of 9% light subunits at pH 7 after short periods at pH 11, and reassociation of only 2 to 4% light subunits at pH 7 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 with phase plate angle 70. a, Soluble protein from third chromatographic cycle on cellulose phosphate, in 0.1 M NapCOa-0.4 M KCI, pH 11.0, at 52,640 rpm for 112 min, 6 mg per ml. b, Precipitate from third chromatographic cycle on cellulose phosphate, dissolved in 0.1 M Na&O$-0.4 M KCI, pH 11.0, at 52,640 rpm for 112 min: upper, 2 mg per ml; lower, 5 mg per ml. c, Myosin dialyzed against sodium phosphate, 0.5 M (upper) and 1.0 M (lower), at pH 8; at 52,640 rpm for 64 min, 8 mg per ml. d, Myosin stored in 0.1 M K&03-0.4 M KCl, pH 10.5, for 20, 3,0, and 1 hours (from the top down) ; at 50,740 rpm for 68 min, 5 mg per ml. e, Myosin st,ored in 0.1 M K&03-0.4 M KCl, pH 10.5, for 30 hours (upper) and 48 hours (lower), then titrated to pH 8; at 52,640 rpm for 96 min, 6 mg per ml. f, Myosin stored in 0.1 M K&0$-0.4 M KCl, pH 11.5, for 20, 3, 0, and 1 hours (from the top down); at 47,660 rpm for 48 min, 9 mg per ml. g, Same as f, at 128 min. h, Same samples 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 except for 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); protein concentration, 10 and 1 mg per ml in each cell; at 52,640 rpm for 88 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, 9 mg per ml. c, Same samples as in b, stored for 5 hours at 4 prior to experiment at 4; at 48,000 rpm for 104 min. d, Myosin stored in 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 guanidine for 5 min; at 48,000 rpm for 228 min, 9 mg per ml. e, Second cycle precipitate from 2 M guanidine-ammonium sulfate fractionation, in 5 M guanidine.IICl-0.4 M KCl-0.001 M EDTA; at 52,000 rpm for 530 min: upper, 10 mg per ml; lower, 7 mg per ml. f, Third cycle precipitate from 5 M guanidine-ammonium sulfate fractionation of carboxymethylated myosin, in 5 M guanidine.HCl-0.4 M KCl0.001 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 M EDTA; 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 M KCl-0.05 M KHzPOh-0.001 M EDTA-0.001 M dithiothreitol, pH 7; at 52,000 rpm for 48 min: upper, 8 mg per ml; lower, 4 mg per ml.

hue

of May

25, 1969

L. C. Gershman, A. Strachu,

and P. Ilreisen
TABLE Time I\

2731

30-40 in 0.4 Y KC1 at, ~1-1 7 indicate dissociation into a light component (-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 earlier stored for 12 hours at 37, Archibald measurements at 8,000 to 13,000 rpm indicate aggregation of the heavy component and, at sedimentation equilibrium at 13,000 rpm, its limiting molecular weight is about 620,000. The data at 40,000 rpm indicate about 6;; of a molecular w+ht 20,000 component and another 4;/u of a molecular weight 90,000 component. Thus, heat treatment of myoairi would appear to result. in di?sociat ion of light subunits from the intact. myosin core and irreversible aggregation of the dissociated components. Light. subunits ma?- also bc dissociated from myosin at guanidine concentrations above.1 to 2 M (Pig. 64, and the full extent of dissociation is attained within the time required for the format,ion of schlieren boundaries (Fig. 64. Sedimentation equilibrium esperiments indicate the dissociation of light subunits, of molecular w-eight. 21,400 and c*omprising 12.0+Zc of the protein, from the intact myosin core, of molecular weight 430,000. On redialysis against 0.4 LI KCl, 1111 7, sedimentation velocity and sediment&on equilibriunl experiments iudicate re:lssociation of the light. subunits with the myosin core, the extent, ranging from two-thirds reassociation after I hour in 2 M guanidine to about one-half reassociation after 24 hours in 2 M guanidine. EJect of Dissocinting Conditions on :I TPnse .4ctitrity of Myoin -. On treatment of myosin above piI 10.0 follon-cd by titration or dialysis to neutral pli, the ATPase activity of myosin is found to be irreversibly lost, the extent of inactivation increasing on prolongtd alkaline trcatmcnt. As shown in Fig. 7 (upper), for myosin in 0.4 M KCl, ATlase in:&vation anti light subunit dissociation occur over comparable ranges in pII. At pI1 10.5 ATPnse inactivation is faster than light subunit dissociation,

dependance of dissocialion and reassociation of light ulknli component Samples 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 KCIpI1 7, for 24 hours. 0.005 M NaHCOa,
Light component Time at pH 11 At pH 11: Sedimentation Velocitya % After dialysis to pII i ~_Scd;nw&;ion Sedimentation equilibriumb __% %

5 1 3 20

min hr hr hr

10.6 12.1 12.0 10.6

4.8 7.5 8.7 8.7

3 .oc 8.0 10.0

a From schliercn patterns obtained within 20 to 60 min of stat,ed times. * Experiments performed at 40,000 rpm; averngc molecular weight of light component is 25,000. c Data at 13,400 rpm indicnte :I molecular weight of 580,000 for the heavy component. and at pII 11.0 :\TPase activity is totally lost within several minutes. Inactivation of ATPase at pl t 11.0 is not prevented by EDTA, dithiot,hreitol, or 31~r++-ATP, nor is activity restored after reduction with 0.2 M fl-mercaptoethanol. However, there protection of ATI&c activity on alkaline appears to be SOIW titration of myosin in 2 M KCl-0.01 JI .%lP (Fig. 7, Io~er), and approximately 10% of coutrol ATIase may be recovered on dialysis to neutral pH after short periods at pII 11.0. The transition zone for light subunit dissociation is broader in 2 M KCYl-0.01 11 ATP than in 0.4 M KCl. The protective effect. of 2 nr KCl-0.01 M ATP n-as utilized to explore the possible relationship between subunit composition and :1Tlasc activity. Myosin at 6 to 8 mg per ml in 2 JI KCl0.01 M AT!? was titrated to pH 11.0 with KgCOa-2 M KCI-0.01 M ATP and salted out with 1 volume of 1.6 to 2.0 u potassium citrate, ~1.1 11.0 (26). Precipitate and supernatant were fractionated by centrifugation at 10,000 rpm for 10 min, and second and third cnyc*le precipitates were obtaiiled by dissolving prccipitate in 0.1 M K&OI-2 M KCl-0.01 JI hTP, pH 11, and repeating citrate fractioilation. Identically treated samples of precipitates, supernntant, and unhactionated protrin were analyzed for ATlase activity after simulta~leous dialysis against 0.4 M KCl0.005 11 NnlIC03, pH 7, and for subunit composit,ion after dialysis 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, unfractionated myosin, whereas enzymic activity is markedly diminished for the Ilart ially purified heavy chain core and is absent. in the light subunits. Experiments were also conducted in which samples of citrate treated but unfractionntcd myosin were diluted with 1 volu~ne of freshly prepared supernntant (containing light subunit,s), 2 volumes of supernatant, or equivalent volumes of On dialysis to ~1-1 7 the 2 11 KCl-0.01 11 XTP-0.1 M K&Oa. solut.ions containing escc,ss light subunits wrre found to have ATlasc activity ap~~rosimatcl~ a-fold greater than the XTPasc activity of identically treated luyosill containing 124 light component.

0.6

t\

3 min

o7

12 1

2 M KCI-0.01

M ATP

10.0

10.5
~ti

II.0

II.5

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

2732
TABLE ATPase V

Subunit

Structure

of h+yosin.

III

Vol.

244, No.

10

after alkalineaclivily and subunit composition citrate ttealmenl and subunit fractionation
Residual light component _.Stdimenta- Sedimentation v&c- tion equilibity riumb

Iraction

ATPase activity@

fin&

Pi/mi?S/l?Sg

7X

Unfrnctionuted 0.100 (ZtO.017) 12.0 11.7 Precipitate ~IIIS one-half supernatant.. . 0.071 (f0.020) Precipitate. 0.052 (f0.009) 5.5 7.2c Second cycle precipitate 0.037 (f0.016) 4.4 Third cycle precipitate. 0.025 (f0.019) 3.9 G.4d Supernatant . 0 100 ~. 0 Average (G.E.) based on five experiments; data are standardized to 20 min of alkaline treatment prior to dialysis. Control ATPase is 0.G fimole of Pi per min per mg. b Experiments performed at 3G,OOO rpm; about two-thirds of t.he residual light subunits are present as a molecular weight 20,ooO component and one-third as an aggregate of molecular weight -90,000. Sulfhydryl groups were not protected in these experiments. c Data at 13,400 rpm indicate a molecular weight of 4.4 X lo6 for myosin core. d Data at 14,000 rpm indicate a molecular weight of 4.5 X lO$ for myosin core.
TABLE Kinetic

VI of myosin
I I I

data on heat denaturcllion

k

as:
SCG-~ 1
83 XW/ 10-e 23 ! 57 2.5 j 53 velocity
E.U.

SEC-
ATPasse innctivation.. Light subunit dissociationa. 4.0 x 10-K .

108

1.1 x 10-s : 8.6 x patterns

91 at 4

0 From schlieren after heat treatment.

on sedimentation

Heat treatment of myosin also results in inactivation of ATPase and light subunit dissociation (23), and the kinetics of both phenomena was studied during incubation of myosin at 3040 over 24 hours. Both AIPase inactivation and light subunit dissociation follow first, order kinet,ics, with rate constants for ATPase inactivation greater than for subunit dissociation (Table VI). Values of activation enthalpy are about 55 kcal per mole for both processes, while the activation entropy is slightly greater for ;YlPase inactivation than for subunit dissociation (Table VI). On treat.ment in 1 31 to 2 Y guanidine, myosin showed no ATPase activity on furt.her dialysis against 0.4 Y KCl, pH 7; nor was ATPase preserved in the presence of EDT.%, dithiothreitol, or ATP. Heavy Subunits of Myosin-The light subunits may be fractionated from the myosin core by alkaline dissociation followed by dilution in water al neut.ral pH (2); after three such cycles the proportion of light alkali component is reduced to 1 to 2% of the

protein (References 2 and 3 and Table VII). On dialysis of third cycle precipitate from reduced and carbosymethylated myosin against 5 M guanidine. HCl-0.4 M KCl, sedimentation equilibrium experimcnt,s indicate dissociation of t.he myosin core into heavy subunits of molecular weight 2 14,000 (Table VII). In order to avoid possible elrects from alkaline hydrolysis of polypcptide chains, ultracentrifugal st.udies were also conducted on the myosin core fractionated iu guauidiuc solution. After two or three cycles of subunit. dissociation in 2 Y or 5 M gunnidine and salting out with ammonium sulfate or potassium citrate (see Methods), the residual precipitate fraction was dissolved in aud dialyzed against; 5 M guanidine~HCl-0.4 M KU0.001 JI EDTA, 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 myosin in 5 u guanidine (I, 3), there is less trailing behind the main component, reflecting the removal of light subunits; an additional leading edge arises from heavy chain aggregation (Fig. 6, f and which USILS 2 M guanidine and potaGum 8). The procedure c&rule provided the best fractionation, and the proportion of light subunits ~a.5 here reduced to 1 to 2c/b (Table VII). Sedimentation equilibrium data indicate liuear plots of log J against 9, yielding a molecular weight of 211,000 for the heavy subunits (Table VII). Comparable vulues are obtained for mgosin aud carboxymethylated rnyosin. Previous studies (41) on myosin have suggested that the heavy subunits are dissociated only at guunidine concentrations approaching 5 M, and this conclusion is confirmed in a sedimen&ion equilibrium esperiment on third cycle precipitate in 4 M guanidine, indicating a molecular weight of 414,000 for t.he purified myosin core (Table VII). Experiments were conducted on reconst.itution of the heavy chain core after subunit dissociat.ion in 5 Y guanidine. On dialysis of guanidine-treated third cycle precipitate against 0.4 M KCl-0.05 N KH2P0,-0.001 Y dithiothreitol, pI1 7, sedimentation velocity experiment.s show a single self-sharpened boundary (Big. Gh), with extensive aggregation indicated by MI 11 S sedimentation coefficient and marked boundary asymmetry. Sedimentation equilibrium experiments indicate reassociation of the heavy subunits into soluble n-mers, with minimum molecular weight. on the order of l,lOO,OOO (Table VII). In similar experiments on the reconstitution of mgosin disociated in 5 M guanidine but not fractionated into light and heavy subuuits, sedimentation velocit,y experiments indicate a main boundary light component. On with s!j, w -8 S and about 3yo r&dual sedimentation equilibrium (Table VII), the reconstituted myosin has a minimum molecular weight, of about 580,000; the residual light subunits undergo marked aggregation in the absence of 0.001 .\I dithiothreitol. COOH-k~minal ilnalyses-SSamples of myosin and partially purified heavy chain core obtained by alkaline fractionation show isoleucine ZE the predominant COOH-terminal amino acid and submolar quantities of several ot.hcr amino acids. In contrast, the purified heavy chain core obtained by means of five cycles of 2 M guanidine-citrate fractionation shows only trace amounts of several amino acids, even after digestion for 3 hours with carboxypeptidase A.
DISCUSSIOS

The low speed sedimentation equilibrium data and the Archibald measurements during early ccnt.rifugation indicate a weight

Issue

of May

25, 1969

L. C. Gemhman, A. Stracher, and P. llreizen

TABLE

2733

1.11
and reconstitution of heavy chain core

Sedimentation

equilibrium

experimenls

on dissociation

All guanidinc EDTA.

solutions

contain

guanidinc.IICI-0.4

M KCI,

pII 5.6; solutions

Eractionation

procedure

totor speed -._ rpm

used for guanidine-salt

fractionation

also contain

0.001 M

Residual

light

component

I
%
3.5 3.5 3.5 2.2 2.2 2.6 2.6 2.6 2.6 4.8 4.8 5.6 5.6 3.6 3.6 1.2 1.2 1.2 2.6 2.6
.J

Heavy

component0 mol Wf x 10-r

nwl rut

In 4

guanidine: third nium sulfate

cycle precipitate,

2 M guanidine-ammo-

36,990 24,630 19,160 36,990 21,720 36,960 20,380 36,160 20,120

18,700 18,709 18,706 20,060 20,000 21,000 21,000 19,500 19,500 20,200 (zk700) 19,300 19,300 18,600 18,600 2QP.500 20,500 2Q,m m,600 20,600 20,200 20,200 19,!100 (~1~800) 45,600 45,600 45,609

(24.2)
14.5 (28.7) 9.03 (23.7) 7.78 7.59 2.14 (22.5) 9.08 5.98 S.68 (25.5) 10.7 6.42

(4.17) 4.14

In 5 M guanidine Third cycle precipitate,* Third Third cycle precipitate,6 cycle precipitate,b

pi1 11, low ionic strength pH 11, low ionic strength pH 11, low ionic strength

(2.4)
2.16

(2.0)
2.11 2.14 (~1~0.025) (1.9) 2.08 2.08 2.07

Average (Gi.D.) Second cycle precipitate, Third Third Third cycle precipitate,c cycle precipitatc,e cycle precipitate,

2 JI guatlidirle-amnlonium 5 M guanidine-ammonium 5
M

sulfate sulfate sulfate

guanidine-ammonium

2 JI grlanidine-citrate

Third

cycle precipitate,

guanidine-citrate

36,180 22,020 36,OCU 18,000 37,010 21,740 36,000 24,000 18,000 37,020 21,740

(2.2)
2.10 2.21 (1.9) 2.14 (rkO.054)

W.6)
8.98 2.11

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

Third Myosin hIyosin

cycle precipitate,d

guanidine-citrate

36,000 13,090 8,ooo 14,290 9,340 36,990 13,410 36,000 14,000

38,600 38,609 19,800 19,800

0.4 0.4 0.4 0 0 3.9 3.9 5.1 5.1

(27.0) 9.04 (35.3) 12.3 13.2 13.9

(12.8) 11.3 (14.0) 11.3 5.9 5.7

a Data obtained at high (T, indicated in parentheses, are not included 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) for rabbit skeletal myosin, in accord with previous weight average values determined 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 preparations of myosin (Table II) and t.heir formation during prolonged centrifugation (Fig. 3) sugbests that the molecular weight of myosin is less than 520,000. Monomer may be resolved from low n-mers on high speed sedimentation equilibrium, and the resulting data indicate a molecular weight of 468,000 (~10,000) for myosin. That these experiments measure monomer n-eight is indicated by the invariance in results for at least 24 hours beyond equilibrium time and at rotor speeds over a range in Q The 468,000 value is in close agreement from 6 to 13 (Table I). with the number average molecular weight of 470,COO (~10,000) reported by Tonomura, -%ppel, and Morales (i) from osmotic pressure measurements on myosin preparations not containing free contnmiuants in significant proportion. -4 molecular weight

in averages.

of 510,000 has also been determined on high speed sedimentat.ion equilibrium (9); the experiments were performed at a comparatively low rotor speed (a - 4), however, and it seems possible that a small proportion of myosin dimers, prment originally or formed during 51 to 118 hours of centrifugation, may not have been fully resolved from monomer. The value for s:,,~ of 6.1 S (Fig. 1) is at the lower end of the range from 6.0 to 6.43 S earlier reported for the sedimentation coefficient of rabbit skeletal myosin (4, 8, 9, 42, 43). Although the presence of a small proportion of low n-mers do= not appreciably affect the sharp schlieren peaks obtained on sedimentation of myosin, preliminary experiments by M. S. Drapkin, with the use of scanner tracings at 2800 A, do indicate rapidly sedimenting material in t.he plateau region; this component is proportionately much smaller than a similar fraction noted on interference patterns of sedimenting chicken breast myosin (44). The over-all findings on the aggregation of myosin in 0.4 x KCl-0.05 M phosphate, pIT 6.5, are in accord with earlier obser-

2734
pH 11 2 M guanidine

Subunit

Stmcture

of Jlyosin.

II1

101. 244, Ko. 10

5 M guanidine

--

I1

5 M guanidine

,z:

FIG. 8. Diagrammatic summary of subunit structure and interactions of rabbit skeletal myosin. The stoichiomctric dat.a indicate 2.7 (M.3) light subunits per myosin molecule, with some evidence for selective association of 2.0 (f0.3) light subunits. Reassociation of light subunits is diminished on prolonged alkaline or guanidine treatment., an11 does not occur after heat dcnaturation. vations. In the most thoroughstudy onsl)olltnlleous:~ggregration of myosin, Lowey and Holtzer (45) concluded that the phenomenon may involve a rate-limiting struct.ural change followed by If higher aggregates may bc neglected, this rapid dimerization interpretation 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 monomer The low speed sedimentation equilibrium weight, and 1 is time. data (Fig. 3) are in agreement with this model and iudicute a rate addition, the observed constant, L, of 1.3 x 10e6 see-I, al 4. 111 aggregation of cnrboxymcthylated myosin confirms the earlier conclusion (45) t.hat, intermolecular tlisulfidc bridges necul not be implicated in the spontaneous aggregation of myosin. However, some kind of covsleut change appears likely, since aggregation is not reversible at 4 sft,er prior storage at 30-40. The preseut data indicate molecular weights of 420,000 for the Previous heavy chain core and 212,000 for each heavy subunit. findings of homogeneity on ncrylamide gel elcctrophorcsis (46) and data on sulfhydryl pcptides (47, 48) would indicate that the heavy subunits of myosin WC similar if not identical, aud this conclusion is supported by the invarinncc in hc:tvy chaiu weight at u values from 6 to 29 (Table VII). The absence of any evident COOH-terminal end group for the heavy chains suggests a cyclical structure or an inaccessible amino acid. Alternatively, the occurrence of several end groups in submolar ratio would be consistent with microhct~erogerleity of the heavy chains, and this iutcrpretation appears to be favored in more recent experiments? The light subunits arc 20,200 in average molecular weight,3 PA. Stracher, unpuhlishcd data. a In a recent study on the purified light alkali componeiit of myosin, lrederiksen and IIoItzer (49) have reported molecular weight values of 33,600 from light scattering - mcasurcments in 0.3 M KCI-0.1 JI phosphate, pH 8, and 31,600 from viscosity measurcments in 5 XI guanidine.HCl. The exneriments were done without protection ocsulfhydryl groups, except for the use of 0.1 M & mercaptocthanol in some of the grianidine experiments. The results arc appreciably higher than the 20,000 value determined from high speed sedimentation equilibrium data on light alkali component (References 2 untl 3 and Table III) or acetylated light component (27). In evaluating the discrepancy in results, it should be noted that the light component readilv undergoes aggregation and, in the absence of sulfhvdrvl protectfion, as much as one-third of the light component may aggregate to the 60,000 to 100,660 weight lcvcl (Tables IV and V). Aeereeation to this cxtent.would result in weight average vr~lr& of 2&06i to 45,000 for It is unlikely that heterogeneity of this kind t.he light subunits.

and comprise 11.7ci;, (&I 7;) of myosin prepared by the SzentGyorgyi method or chrolllatogral)hed once on DEAE-cellu1ose or cellulose phosphate. These data indicate 2.7 (~0.3) light subunits per myosin molecule (Fig. 8). In a somewhat less precise calculation, t.he diffcreuce iu molecular weight between myosiu aud heavy chain core would indicate :I total weight of approximately 50,000 for the light COII~~OIIC~~, that is, about 2.5 light subunits per myosiu molecule. The stoichiomctric data must be iuterprcted with caution, however, since Locker and Hagyard have reported quantitatively differeut electrophoretic bands for the light subuuits isolated from rabbit skeletal and cardiac myosin (50) and red uud white skclctal muscle (51), raising the possibility of Illicrohetcrogetlrit~ with two or three light subunits per myosiu molecule. There is also evideuce that two light subunits may be sclecThe light, component tivcly associated with the heavy subunits. has been earlier identified in heavy meromyosin (2, 27, 52), nud heavy meromyosiu IIMJ- be degraded into two particles of Subfragmeut 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 cellulose phosphate chromatography (Table III) the residual soluble or 2.0 myosiu contains oiilg 8 lo 3;; of light alkali component, If this finding may (~tO.3) light subuuits per myosiii molecule. be extrapolated to the bulk of dcnnturcd protein, it would appear t.hat, some of the light alkali component may be less tightly bound be a11 essential part of t.o t,hc heavy chain core or 111ny nut CWII myosin. In this regard myosin exhibits variable amouuts of 5-adenylic acid deaminase (22, 26, 34, 44) and myokiuase (26, 44) activities; however, neither of these IWO proteins rcpresents a major fraction of the light alkali romponeut, for on purific:ltion from rabbit skeletal muscle 5-adenylic dctlmiuase has a scdimemat ion coefficient of 12 8 (56) :tnd myokinase has different sulfhydryl pept ides (18) aud COOH-termiual group (57) than the light, subunits of myosin. On the preyent evidence it would thus appear that rabbit. skrletal myosin contains two light subunits ( -9y0) with some certainty, and additional low molecular weight, material (2 to 37;) that may possibly reprrsent a less tightly bound light subunit, Irlicrohetcrogerleit~ of myosin, or trtcc coiitan~iiiaiits . . With respect to the rcliort of Gaetjens et al. (26) that the proportion of light subunits is reduced to 4.8):, aftrr chromatography on IN%E-cellulose and cellulose phosphate, it should be noted that the 3.8y0 value reprericnts the recovrred yield of light subunits on their purificnt.ion (26), and is not. based on direct ult,raccutrifugal analysis of the entire myosin prcparatioii. Significantly, ullchrornntogrnl~hed myosin was found to yield only 3.2L; of light subunits (26), a proportion even less than that determined for twice chrorn:ttogral,hed myosin. Also, the published schlicreu patterns (26) cm myosin aud twice chromatographed myosin show comparable proport ions of light component on succinylnt.ion. That t.he 4.8c/, vnhe may be attributed to incomplete recovery of light subunits rather than to chromntogrnl)hic purification would be consistent with previous reports would be discerned from the angular dependence of light scattering measuremcnt,s, since the calculated dissymmetry is only 1.01 for a sphere of 166.4 diameter or a cylinder of 156 A length (very crudely approximating an n-mer of molecular weight 100,000). In any case, light scattering and viscosity measurements on an aggregated syst.em are clearly referable to an average value, and one may qiiestion whether the higher molecular weight vtl~ms (40) describe light chains that, arc in part aggregated.

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 solubility methods is apl)reciably 1~5s than the proportion detcrmincd on ultracrntrifugal analysis. The proposed model for native myosin (Fig. 8) indicates a weight average molecular weight of 18i,OOO IO 190,000 on complete dissociation into light and heavy polypeptide chains. This value may be compared with the weight average molecular weight of 19i,C@O that, was determined by Woods, Himmclfarb, and IIarritlgtoll (1 I) from low speed sedimentation equilibrium experiments on mywin in 5 JI guanidinc. HCl. These esperimcnts (11) were contlucled on myosin that ~;II; unfractionatcd with respect to light wbuuits and ~)rcsum:tbly contained most if not all of the light component of native myosin. In support of this interpretation, it. has been noted earlier (3, 12) t.hat :\rchihald experiment.s (I 0) and sedimentation velocity esperimerits (46) on identical lxeparations of myosin are suggestive of low molecular weight hetcrogelwity in gunnidiue and urea solutions. The dissociat.ioll of light subunits at l)I-I I1 and in 2 JI guanidine awonipanies r:il)itlly owurring struct.ur:il changes in myosin, manifest in bot,h (*a~: by inrreaae in levorotat ion and decrease ill intrinsic viscosity (-II, 58). These changes presumably involve an unfolding of the helicul conforni:ltion in niyosiii (-II), although some of the fall in intrinsic viscosity is attributable to subunit dissori:it.ioii. I )wl)ite nearly comp1t:t.s unfolding in 3 to 4 M guanidino (1 I), the heavy chain core remains intact at guatiitliile coiiwntratiolls up t.0 4 M. .-\ftcr light subunit di>so11 guanidine, at least two-thirds of the ciatioii at: 111 11 or in 2 1 light subunit:: reawwiatc with the he:lvy chain core on subs~ qiient dialysis ag:lin+t 0.4 XI KCI, l)t 1 7 (Fig. 8). Complete rm?;sociat ion al)pwrs to be prevent cd by changer, possibly covalent Fide chaitl re:tct.iow, that occur OJ~ prolotlged alkaline or gwnidinc trcat.nncnt, a11t1 in the c:we of heat dennturation there is no reaxsoc:iat.ion at all. Light subunits tend to aggregate ill the absenw of sulfhydr>.l lxotection; howvcr, subunit rcassociation is not. augnwnlcrl in the lnwtwcc of 0.001. M dithiothrcitol or after prior carhosyinct hylation of niyosin. The present data confirm the report (-II) that. the heavy chain core tnay br rwonst ituteti after dissociation in 5 M guanidine that aggregation to low (Fig. i), :11x1 thrrc is some ovitlctw n-incrs is cwtiallccd in the abs;cnc,c of light wbunil~. While guauidinc-i rratccl rn\oGn 111x?- be rcconst itutcd with at least some of the protein at, the 500,000 to 600,000 weight. lcvcl (Referenw 41 and Table VI I), the guaJlidilic~-tlc~ltctl but fractionated heavy subunits arc rcronstitutetl with ;I mil~irnuni weight on the order of I ,100,oW (lal)lr TII). The purifietl heavy chain core aggrcg:itcs to :I >imil:ir lcvc~l after the rcnwv>il of light subunits by alkaline fr:ic:tioi~al ion (2, 3). Purttirrniorc, rrconst itution cslwiment~ on g~J:lllitliJlr-trc,Rted nlyosin have indicated extcnsivc aggregnt ioll inwIving intcrmolrcwlar tlisulfidc bridges (41); howwr, the I~e:lvy chain core undrrgocs :~ggrcgation in 0.4 M KCI at pH 7 after lwior retluct ion and c~:lrt)osrJleth:lutioJl (2, 3), in 0.001 M dithiothrcilol (Table VII), :IJI~ without sulfhydryl rlw-;t. that removal of l)rotwt.ion (T:ibIe VII). The fintlings >u,, light subunit> rna\~ uncover -ites on the hexvy c+~:lins that enter into 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 subThe iiliits arc involved in the biological activity of nlyosin. dissociation of light. subunits from the l1wv.v chain core at plI

11; in 2 31 guanidiue, a11d 011 heat treatment is accompanied by the inactivation of .\TPase, indicating that both .\TPase inactivation and light subunit dissociation may follow common The values of AHI and AS: for both dcnnt wing procwsrs. phenomena are comparable with values reported 011 heat denatrwtion of other proteins (59), and the more rapid rate for Allasc inactivation is maniftst by a slightly grcatrr activation entropy for subunit dissociation. That subunit interactions and hLPase activity may be more essentially related is further suggested by the esperiments involving alkaline dcnaturation of Il1yosin in 2 ar KCl-0.01 11 .YIl (Table T). There appear-r; to be r;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 on The results addition of freshly dissociated light subunits. suggest, that the ATPax activity of myosin may involve some kind of interaction between light and heavy subunits, and the question will be further esl~lorcd in a subsequent comniunical.ion OJI the lwolwties of myosin in concentrated salt solutions. lGn:~lly, we may conxirler whether the light ant1 heavy polylwl)tidc chains of JIU~ ivc myosin belong to a single molecule or whether native mywin comprises several clowly associated but distinct ~noleculcs. The present el-idencr would appew to favor a structure of two axially symmetric, eiizymiwlly active protomers, 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 heavy subunits in molar ratio in each particle of Subfragment. 1 (32), the Ftoichiometric presence of two light subunits aft or wpeuted cctllulor;~: pho~l)hate chromatogral)hy of mywin (Table III), the clwe relationship belwccn light subunit dirsnriat ion and irrc~waible .iTPasc in:wti\-xtion (Refrrcnw 40, Fig. 7, and Table VI), the dependenw of partially rrgcneratctl .Yllase activity on subunit composition after alkaline trcatmcnt in 2 M KCI-0.01 M ATP (Table V), and the marked agjircgation of the purified heavy chain core in 0.4 M KU, pH i. Scverthclcss, it would be premat.ure to reach a final conclusion on this evidence alone, :111dthe above hypothesis is under active inw<t igation. ;Ic~,~o2cle~gl,lentsWC gratefully acknowMgc the trchnical :wsist:incc of Miss %eiinid:i L:~~~ixina, JIr. 1)eimis Richxds, 1Iiss Laura Lilx+s, niltl Mr. i\I:ick Errca. We thank I)r. 11. 1Grb11y and colleagues for the ol)l)ortunity of wading their manusrript (26) prior 1.0 publication.

. (;EILSHM;\N,
rlcnd. sci.

f,. c., ~hU3ZEX. I., .\Sl) 8Il:.\CIl,?I~, u. s. .I., 66, 9(X (19f0).

;I.,

t,X.

.\-OL.

R. I)REIZEN, I., (;ERSJJW\N, I.,. C., TIWTT.\, 1. P., WI) S.rIc.wIIEIf, A., J. Gen. Physiol., 60, 85 (1067). 4. IIoLTzE:R, A., .\sn I~nww, s., .I. :lfiz. (:hcw. &x., 81, 1370
(1050). --

( I*rcderiksen and IIoltzcr (4!)) have recently reported the rctcovcry of X to 80%; hTI-:lsct wtivity after sul)lulit dissociation of myosin at plf 11, storage at plT 11 for lOOmin, and Lwk titration to pH 7. It. should he noted that comparnhlc nlk:llinc~ treatment OF mywin hns twrn cnrlior wported to reslllt in the tot:11 loss of Allase activity (3, 21, 21i, 58), and the present, c~spcrimcnts indiratc recovery of 50% ATP~Lw activity, or gro:tt(~r, only after rnrlch shorter periods at pII less than pIl 10.5, a c*ondition under which the dissociation of light component is negligible (Fig. 7, uppw). The reasnn for the tlisrrcpancy is unclwr. s 1,. C. Gershman, and P. Drtrizen, manuscript in prop:w:ition.

2736

Subunit

Structure

of Myosin.

III

Vol. 244, So. 10

5. ITOLTZEX, A., LO~EY, S., ASD SCHUSTER, T. XI., ilfokcular basis 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., Riochemistry, 6,3154 (1967). 10. KIELLEY, W. W., ,\ND I~ARRIN(;TON, W. E., Z?iochim. Biophys. Acta, 41, 401 (i960). 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,I<. N., ASD ~IITCFIELL, E. H., Arch. Biochem. Hiophys., 79, 191 (1959). 15. CONNEI~L, J. J., Atin ALCOTT, H. S., Arch. Biochem. Biophys., 94, 128 (1961). 16. LOCKER, R. II., AND IIAGY.IRD, C. J., Arch. Biochem. Biophys., 120, 241 (1967). 17. RI~HAIUU, E. G., CIIUNG, C. S., AP~EI,, P., ASD OICOTT, II. S., Fed. Proc., 28, 727 (1967). 18. WEEDS, A. G., Biochem. .I., 106, 2X (19G7). Slruclure 19. SZENT-GY~jIXYI, A. G., in C;. H. R~URNE (Editor), und fun&ion of muscle, Vol. ZZ, Academic Press, New York, 1960, p. 1. 20. WETLAUFER, D. H., ASD Ens,\Lr+ J. 1., Biochim. Riophys. A&a, 43, 132 (1960). 21. GERSHM.~S, I,. C., STRICIIER, A., .tsn ~~I~EI~ES, P., in W. Cr. CHEWTHER (Editor), Symposium on jibrous proteins, 1967, Butterworth and Company, I,td., Sydney, Australia, 1968, p. 150. 22. LOCKER, 11. II., Biochim. Biophys. Acla, 20. 514 (1956). 23. YUXI, T., HI\SIIIMOTO, Y., AND TONOMUR.~, Y., Arch. Z?iothem. Hiophys., 87, 55 (1960). 24. OPPESHEIMER, II., I%~I&NY, K., I~AMOIIL, G., -4~1) FENTOS, J., Arch. Biochem. Biophys., 120, 108 (1967). 25. SRETEI~, 1. A., J. Gen. Physiol., 60, 116 (1967). 26. GAETJESS, E., B.&H~NY, K., BAILIN, G., OPI~ENHEIMRR, II., AND BUNNY, M., Arch. Biochem. Biophys., 123,82 (1968). 27. LOCKER, R. H., AND HI\~~l\~~, C. J., Arch. Biochem. Uiophys., 120, 454 (1967). 28. HARTSHORNE, D. J., ASI) STRACHRR, A., Biochm. Z., 346, 70 (1966). 29. II.\RRIs, M., .IND SIW~IEH, H. C., Biochim. Biophys. Acfa, 133, 393 (1967). 30. LOCKER, It. I-I., Biochim. Biophys. A&a, 14, 533 (1954). 31. SARNO, J., '~ARENDASH, A., AND STR.~CHEI~, .4., Arch. Biochem. Biophys., 112. 378 (1965).

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