ERICELLENBOGEN,~RAJA IYEKGAR,HOWARDSTERN,$AND ROBERT E. OLSON
From the Department of Biochemistry and Nutrition, Graduate School
of Public Health, University of Pittsburgh, Pittsburgh,
Pennsylvania
(Received for publication, April 11, 1960)
Although many physicochemical studies of skeletal myosin have been
reported (l-4), comparatively few comparable studies have been made
of cardiac myosin (5-7). The assumption that the myosins derived
from skeletal, smooth, and cardiac muscle are identical (8) is not
based upon any single rigorous compara- tive study.
This laboratory is presently devoted to a systematic study of
myocardial metabolism in dogs and man under various condi- tions of
health and disease (9). The present communication is devoted to the
physicochemical characterization of myosin iso- lated from the
hearts of normal dogs. Preliminary reports of this work have been
published elsewhere (10, 11).
EXPERIMENTAL PROCEDURE
Zsoldion of Myosin-Healthy, normal dogs were anesthetized with
Nembutal(25 mg per kg) and a thoracotomy was performed. Respiration
was maintained by intermittent oxygen under posi- tive pressure.
Only those animals with normal cardiac function as ascertained by
normal heart size, normal sinus rhythm, normal electrocardiogram,
and normal arterial and venous pressures were used in this study.
After opening the pericardium, each heart was excised by rapid
transection of the great vessels, and dropped into iced deionized
water. Since ventricular fibrillation was found to reduce the yield
of myosin considerably, the con- tinuation of a regular ectopic
ventricular beat for a few seconds in the ice water after excision
of the heart was adopted as an additional criterion of
normality.
In this, as in all succeeding steps, deionized water with a
conductivity not higher than about 1.25 x lo--’ mhos per cm was
employed. The hands of the experimenter were gloved. Fat and
connective tissues were removed while the heart was cooling in the
deionized water, and myosin was extracted ac- cording to the
procedure of Szent-Gyorgyi with some modifica- tions (2). For most
of the experiments described, 90 f 10 g of trimmed heart muscle
were available. The heart tissue was pressed with crushed ice
through a cold meat grinder with holes of 2 mm diameter. To the
cold mince were added 300 ml of a cold phosphate-KC1 buffer (0.3
ionic strength, pH 6.8, phosphate buffer in 0.3 ionic strength KCl,
adjusted to pH 6.8) and the mixture was stirred at 2” for 20
minutes. During this time 10 ml of a 1% solution of the dipotassium
salt of adenosine triphos-
* Supported in part by grants-in-aid from The National Heart
Institute (H-1422), National Institutes of Health, United States
Public Health Service, American Heart Association, New York, and
the Lasdon Foundation, New York.
t Died May 29, 1960. $ United States Public Health Service
Postdoctorate Research
Fellow, 1954 to 1956. Present Address, E. R. Squibb and Sons, New
Brunswick, New Jersey.
phate (KrATP) adjusted to pH 6.8 were added. After stirring, the
mixture was pressed through cheesecloth, diluted with 1200 ml of
cold water, and filtered with slight suction through a Buchner
funnel covered with a layer of shredded filter paper pulp between
two sheets of filter paper. The filtered solution was diluted with
3000 ml of water while being stirred, whereupon the myosin
precipitated out. It was allowed to settle at 0” for at least 3
hours. The clear supernatant fluid was removed by decant&ion
and the precipitate collected by centrifugation. To each centrifuge
tube containing the wet myosin precipitate an equal volume of 2.0 M
potassium chloride adjusted to pH 6.8 was added and the myosin
solutions were combined. The pre- cipitation procedure was repeated
and the wet myosin was dis- solved in an equal volume of 1.0 M
potassium chloride (pH 6.8). Three milliliters of a solution of 1%
Kz-ATP were then added, and an aliquot was analyzed for homogeneity
in the analytical ultracentrifuge. This material is usually called
“crystalline” myosin and produced ultracentrifuge patterns as shown
in Fig. la.
The remainder of the myosin solution was then subjected to
preparative ultracentrifugation at 0” in the analytical ultra-
centrifuge at 39,900 r.p.m. for 2.5 hours. After preparative
ultracentrifugation, the top 8 ml of the 11 ml of solution in each
tube were pooled, diluted with 25 volumes of deionized water, and
the precipitated myosin was collected as before. A volume of 1.2 M
potassium chloride, adjusted to pH 6.8, equal to the volume of the
packed precipitate was then added to each tube. The redissolved
myosin solutions were pooled, 1 ml of 0.3 ionic strength phosphate
buffer (pH 6.8) was added, and another sample was analyzed in the
analytical ultracentrifuge. Often, the preparation at this stage
was found to be homogeneous in the ultracentrifuge (Fig. Id) but if
a slight leading edge was ob- served (Fig. lb, c), the preparative
step was repeated. As soon as an ultracentrifugally homogeneous
preparation of myosin was obtained, it was dialyzed through
cellophane for 3 days against 6 changes of 20 volumes of 0.6 ionic
strength potassium chloride adjusted to pH 6.8. For light
scattering measurements, dialysis was carried out at pH 7.2 (12)
and for electrophoresis it was carried out in 0.1 ionic strength
Verona1 buffer in 0.2 ionic strength potassium chloride at pH 8.3.
Only eshaustively dialyzed solu- tions were accepted for
physicochemical studies. The yields of pure myosin were rather low,
averaging around 300 mg/lOO g of heart, since purity rather than
high yield was the objective.
For comparative purposes, a few preparations of rabbit skeletal
myosin were made by these same methods. Skeletal muscle was
obtained by excision of whole resting muscle from the anesthe-
tized animal. The skeletal myosin obtained in this manner also gave
rise to ultracentrifuge patterns indicative of a homogene- ous
substance.
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September, 1960 E. Ellenbogen, R. Iyengar, H. Stern, and R. E.
Olson 2643
The concentration of myosin in the dialyzed solution was ob- tained
by drying approximately 2 g of solution at 80” until all solvent
was removed, followed by drying in a vacuum at the same temperature
to constant weight. With the dialysates as controls, protein
concentrations were obtained by difference. Dilutions were made by
weighing chosen quantities of stock solution and adding the desired
weight of dialysate. For each preparation, measurements of the
ultraviolet absorption were also carried out. For cardiac myosin,
X,,, in water is 279 rnp and E’s i em = 6.28 f .30 at this wave
length.
Partial Xpeci$c Volumes-Partial specific volumes were deter- mined
by measuring the density of a series of myosin solutions at 1 .O”
in pycnometers with a volume of about 4.5 cc. Weighings were
carried out to five decimal places, with an error of &0.00003
mg, making the partial specific volume accurate to ~1~0.002.
Sedimentation Velocity Xtuclies-Measurements of sedimenta- tion
constants were carried out in a Spinco model E analytical
ultracentrifuge. Solutions above 0.2 % protein were analyzed in the
conventional manner from schlieren patterns. More dilute solutions
were analyzed by means of ultraviolet absorption measurements,
employing the Spinco Analytrol for plotting the densities of the
absorption bands. A few experiments on identi- cal solutions were
carried out in order to determine the effect of the centrifugal
field upon sedimentation constant and boundary spreading. Since no
dependence upon speed was found, all runs were made at the same
speed (56,100 r.p.m.). Sedimentation runs were carried out at first
near 24” and later near 4”. In general, homogeneous solutions of
myosin gave identical sedi- mentation constants (spg,,J independent
of temperature and speed.
Sedimentation-Equilibrium Studies-Sedimentation-equilib- rium
studies were also carried out in a Spinco model E analytical
ultracentrifuge. Dilute myosin solutions (0.2 and 0.1%) were spun
in a double sector cell at 2,994 r.p.m. until equilibrium was
obtained, usually for 7 days. Buffer was placed into one sector
(0.35 ml) and myosin (0.2 ml) was layered onto Dow-Corning fluid
703 (0.1 ml) in the other sector. By means of the constant
temperature device, it was possible to control the temperature
throughout these runs to better than 0.05” at a temperature of
2.5”. The more concentrated solutions were studied by means of the
schlieren and ultraviolet absorption methods, and the more dilute
ones by means of the Rayleigh interference and ultraviolet
absorption methods. Total concentrations in the ultracentrifuge
were determined for each method by carrying out runs in the double
sector synthetic boundary cell. 1Molecular weights were calculated
at equilibrium across the cell, as well as during the approach to
equilibrium at the meniscus and bottom. Approach to equilibrium
studies were carried out at a higher speed (3,194 r.p.m.).
Di$usion ikleasurements-Diffusion constants were estimated both
from the boundary spreading observed in the ultracentrifuge and
from free diffusion in the Spinco model H Tiselius electro-
phoresis apparatus.’ For a homogeneous system, these values should
be identical and this was borne out in those preparations in which
both types of measurement were made. Three different solutions were
run simultaneously at 0.9” for 5 days each. Dif- fusion constants
were calculated from schlieren patterns and from Rayleigh fringes,
employing the method of second moments.
1 We are indebted to Dr. M. A. Lauffer for a single determina- tion
and to Dr. William R. Merchant for making available to us his
electrophoresis instrument for a number of other measure- ments
.
FIG. 1. Sedimentation patterns of various normal dog heart myosin
preparations. Speed 56,100 r.p.m., 4-g”, 0.6 1~ KCl, ad- justed to
pH 6.8, 16.minute intervals from left to right. (a) “crystalline”
myosin; (b) “crystalline” myosin after first pre- parative
ultracentrifugation-note fast component (actomyosin); (c) same
a,fter second ultracentrifugation-note lesser amount of fast
component; (d) same after third preparative ultracentrifuga-
tion-note absence of fast component; (e) pooled bottom frac- tions
from preparative ultracentrifugations after treatment with ATP to
decompose actomyosin, followed by dilution with water- note small
amount of fast component and good recovery of myo- sin; (f) pure
myosin, 2 mg per ml; (g) same as (f) but diluted to 1 mg per
ml-taken at the same angle to illustrate increased boundary
spreading.
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0 0.1 a2 0.3 0.4 0.5 0.6 0.7 0.9 0.9 gm/lOOml
FIG. 2. Sedimentation constants of normal dog heart myosin as a
function of concentration. 56,100 r.p.m., T = 4-7”, 0.6 M KCI, pH
6.8.
2 z
gm/lOOml
FIG. 3. Diffusion and boundary spreading constants of normal dog
heart myosin as a function of concentration. 0.9”, 0.6 M KCl, pH
6.8.
In the most dilute solutions, only 3 Rayleigh fringes were avail-
able for computation, and it is felt that the value for the most
dilute solution might be in error by as much as 10%. This procedure
allowed measurements on solutions of six different concentrations
within 2 weeks after the isolation of myosin.
Limiting Viscosity Number-The limiting viscosity number was
determined on all solutions on which partial specific volumes were
obtained. An Ostwald viscometer with a water time of about 180
seconds was mounted kinematically in an unsilvered Dewar flask. The
temperature inside the Dewar flask was regulated by keeping ice in
equilibrium with cold water, and the Dewar 5sk itself was placed
into a 15-gallon cold bath at 1.0’ in a walk-in cold room kept at
2”. Triplicate determinations were obtained with a maximal
deviation of ~0.3 second.
Light Scu&ri?zg Measurements-Light scattering measurements were
carried out in a &ice-Phoenix light scattering photometer
equipped with a Brown recorder as detector and calibrated by the
manufacturer. The wave length chosen was the mercury
* Recommended nomenclature by International TJnion of Chemistry, J.
Polymer Sci., 8, 257 (1952). This is identical with “inkinsic
viscosity,” a term still widely used.
blue: line (436 mp). ‘l’hc refractive index increment was deter-
mined at the same wave length in a Brice-Phoenix interference
refractometer at 20” and was found to bo 0.206 on a weight frac-
tion basis. With the rclcommrndations of Rupp (12), measure- mcnts
\vcre rarried out at temperatures of less than 15”. Three methods
of clarification were employed: pressure filtration (under
nitrogen) through ultrafine sin&cd glass filters, pressure
filtra- tion through fine sintered glass filters, and
ccntrifugation at 3” in the analytical ultracentrifuge in a
swinging bucket rotor at 39,900 r.p.m. for 30 minutes. Since no
significant difference was observed among these three methods in
the scat.tering of the solvent, the last method of clarification
was adopted. At first measurements werr curried out from 45” to
135” at 15’ intervals in cylindrical cells with 11 ml of solution,
but sincr the Zimm (13) plots went through a maximum at 90”,
succeeding measure- ments were carried out in a semioctagonal ccl1
at 0”, 45”, 90”, and 135” only. The crlls w(lre first thoroughly
cleansed with deter- gent, rinsed several timcls with deionized
water, and were then coated inside and outside with “DcsicotC.”
Before each meas- uremcnt, the coated cells were rinsed with
detergent, thoroughly rinsrd with deionized water and solvent,
followed by a rinse with clarified solvent. The clarified protclin
solution was then placed into the cells and the ~11s w(Ar(: coverrd
with a glass lid. At each angle, a minimum of five readings were
made and protein concentrations were determined on the solutions
afterwards. The scattering due to the solvent never escceded 15c/,
of the scattering of the most dilute protein solution.
Rlectropkureti-Electrophorcttic studies were carried out in the
Spinro model II clcctrophoresis-diffusion apparatus. Mcas- uremcnts
wcrc mado at 4 milliamperes and 49 volts in the micro-
rlcctrophoresis cell, and mobilities were determined from the
schlirrm as wrll as from the Rayleigh fringe patterns. TJnder these
conditions, no evidence of heterogeneity was observed.
A TPase nctitity-The ATPase activity was determined at 25” on
samples dialyzing against Verona1 buffer (pH 8.6) or glycine buffer
(pH 9.2) in order to remove all phosphatt: ions. The method of
Gergely (14) was employed, as well as a modification of his
procedure, with Verona1 buffer at pH 8.6. Time studies confirmed
that the initial rate was linear for the first 5 minutes. A’l’Pasc
activity is expressed as &r, i.e. ~1 of phosphorus liberated
per mg of myosin per hour.
RESULTS L
The results of the physicochrmical studies outlined above are shown
in Figs. 2, 3, 4, 5, and 6 and are summarized in Table I.
The points in Fig. 2 show the dependence of the sedimentation
constant of normal canine cardiac myosin on protein
conccntra-
O-d2 ‘M
Qnfqm I moo gmlpm .I000
FIG. 4. T,ight scattering and distiymmctry measurements on normal
dog heart myosin as a function of concentration. 12-15”, 0.6 M KCI,
pH 7.2.
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I 0 al a2 a3 a4 0.5 a6 0.7 08 a9
gm/ tooml
Fro. 5. Viscosity measurements [(l/c) In (&o)] on normal dog
myosin aa a function of concentration, l”, 0.6 M KCl, pH 6.8.
tion for 8 normal animals. The concentration dependence is linear,
and the solid line was computed for all points by the method of
least squares. At zero protein concentration, the mean
sedimentation constant, SOW,~ for all animals was 6.12 S. The
slopes of the sedimentation plots for each individual dog closely
paralleled one another. The s’&,, value for the indi- vidual
dogs ranged from 5.60 to 6.69 with a mean of 6.16 i 0.13 (s.e.“)
and the slopes (-da@) ranged from 2.2 to 3.6 with a mean of 3.10 f
0.16 (s.e.).
The results of free diffusion and boundary spreading measure- ments
are shown in Fig. 3. The concentration dependence is striking and
nonlinear. The boundary spreading coefficients, calculated from
velocity sedimentation experiments and cor- rected for the
concentration dependence of the sedimentation constant (15), agree
well with the values obtained from the free diiusion studies. Had
the correction for the concentration dependence of s been omitted,
the boundary spreading coef- ficients would have been lower than
the free diiusion coefficients by approximately 20%. Had the
solutions not contained a homogeneous protein, furthermore, it
would not have been possible to compute boundary spreading
coefficients from sedi- mentation velocity experiments, since they
would not have been independent of the time of sedimentation. Upon
extrapolation of the best line to zero protein concentration (Fig.
3) the value for the diffusion coefficient was found to be 2.46 x
lo-’ cm*/sec.
The partial specific volume of cardiac myosin was found to be
0.731. As shown in Table I, the molecular weight of cardiac myosin
calculated from &I,~, Do2o,o, and r? was 226,900. Control
preparations of skeletal myosin from rabbit psoas muscle had the
following constants: a$+ = 6.10, L&J,, - 1.05 x lo-’ cm*/sec
(from boundary spreading); 0 = 0.740. Calculation of the molecular
weight of skeletal myosin from these data gave a value of 540,000
in reasonably good agreement with values re- ported from other
laboratories (16-19).
The molecular weight of cardiac myosin obtained from velocity
sedimentation and diffusion measurements was next checked by the
independent method of equilibrium sedimentation. The results
obtained in four sets of experiments are listed in Table II. The
homogeneity of our preparation was evidenced by the fact that
essentially identical values (within 2yJ were obtained.
s se. = standard error.
‘I I I I
0 0.1 02 0.3 0.4 0.6 0.6 0.7 OB 0.9
gm / loom1
FIG 6. Viscosity measurements [(l/c) In (z/m)] on normal rabbit
skeletal myosin, lo, 0.6 M KCI, pH 6.8.
TABLE I
Characterization of normal dog heart myosin
s”20.w de/de (g/100 ml) DO 2o.w li hl M (from e,D) f/f0 (from M,s)
f/f0 (from M,D) f/f0 (from 0) a/b (from above) M (equilibrium
sedimentation) p (from M,s,B) a/b (from above) ,Y (from D) a/b
(from above) M (from light scattering) length width ATPase
activit.y mobility (pH 8.3, I’/2 =I 0.1 Ver- onal + 0.2 KCI)
6.16 s -3.10
2.46 X 10-7 cm)/sec 0.731 50 c.g.s. units m,ooO 2.15 2.14 2.14 24
222,000 f 4,000 2.77 X 10’ 29 2.71 X 10’ 27 270,000 690 A 28A 350/A
P/mg/hr
- 2.11 cm*/volt/sec
The molecular weights were independent of the nature of the
experiment (approach or true equilibrium), were independent of the
position in the cell at which calculations were carried out (top,
bottom, across the cell), and were independent of concen- tration
and method of optical analysis. The mean value of 222,000 obtained
by this method is in good agreement with the value calculated from
solo,,, DOto,.. and B as shown in Fig. 1.
The estimates of molecular weight of dog heart myosin from light
scattering measurement are also in reasonable agreement with the
above data. In Fig. 4 are plotted the turbidities (un- corrected
for dissymmetry) at 90” against protein concentration, as well as
the diiymmetry ratios &/RI%, at 45” and 135”, respectively.
Applying the correction factors tabulated by Doty and Steiner (20),
the extrapolated molecular weight of cardiac myosin was found to be
270,000 and the dissymmetry at zero protein concentration 1.312.
The resulting variation in molecu- lar weight from a choice of
molecular model is shown in Table
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TABLE II Sedimentation equilibrium studies
Experiment No.
r.p.m.
3,189 Approach to equilibrium Schlieren
3,189 Approach to equilibrium Schlieren
2,994 At equilibrium Rayleigh interference 2,994 At equilibrium
u.v.* absorption 2,994 At equilibrium U.V. absorption 2,994 At
equilibrium Schlieren 2,994 At equilibrium U.V. absorption 2,994 At
equilibrium U.V. absorption
Molecular weight
Average: 222,300 f 3,900
- * U.V. = ultraviolet
centration from 0 to 0.4 % as evidence of the internal consistency
of the data obtained by two methods. These data taken from the
solid lines in Figs. 3 and 4 are presented in Table IV. The actual
mean ratio of molecular weights estimated by the two methods will
be obtained by dividing (D/s)/(Hc/r) by the quan- tity RT/(l - 5~)
which has a value of 9.03 x 10’0 at 20”. When the average values
for the ratios (D/s)/(Hc/r) in the last two columns of Table IV are
divided by this number the mean ratio of molecular weights over the
range of concentrations studied is 1.04 for the coiled model and
1.03 for the rod. This agreement is regarded as remarkable,
particularly since these parameters are strictly proportional to
the reciprocal of the molecular weight only at infinite
dilution.
The changes in relative viscosity of cardiac myosin solutions as a
function of concentration also suggest d&aggregation of the
protein in very dilute solutions, as shown in Fig. 5. At zero
protein solution the limiting viscosity number extrapolated from
measurements on nine preparations was 50 c.g.s. units. Previous
studies of the concentration-dependence of the viscosity of cardiac
myosin were done with impure preparations at higher concentra-
tions (21) and the sharp downward reflection of the curve at 1 mg
per ml was apparently missed. Study of three preparations of myosin
from rabbit psoas muscle studied over the same con- centration
range gave a zero intercept of 266 c.g.s. units in agree- ment with
other observers, as shown in Fig. 6 (22).
Measurements of ATPase activity of purified cardiac myosin at 25’
gave a Qr of 350 f 34 (s.c.). Impure preparations con- taining
actomyosin gave higher values but all values were lower than that
reported for skeletal myosin (14). This low value for the ATPase
value of cardiac myosin is in accord with similar findings reported
by Gelotte (6), Gergely et al. (21), and Tcnow and Snellman (23).
Cardiac actomyosin threads produced in uitro by combining cardiac
myosin with homologous actin (2) contracted visibly under the
influence of added ATP.
DISCUSSION
Our studies provide a set of physical constants for normal cardiac
myosin (myosin C) under constant environmental condi- tions of
temperature and ionic strength. The preparations, furthermore,
appeared to be homogeneous during both equilib- rium and velocity
ultraccntrifugation and during electrophoresis. Mean estimates of
molecular weight from measurements of velocity, sedimentation,
diffusion, and partial specific volume
TABLE III Choices of size and shape from light scattering
measurements*
Mol. wt. Size
Sphere 265,000 Diameter 665 A Rod 272,000 Length 965 A Coil 279,000
Length 674 A
* The above values were computed by combining the values for He/r
and for the dissymmetry at zero protein concentration.
TABLE IV Internal consistency of estimates of molecular weight
from
sedimentation and diffusion constants and light scattering
measurements at different concentrations
T - I I(~/s)/(ncls)l x lo-lr for (HC/T) x
100
z
~~ g/loo ml
0 0.400 0.05 0.320 0.10 0.250 0.15 0.185 0.20 0.155 0.30 0.132 0.40
0.122
coil rod --
coil rod
3.68 3.64 0.109 0.110 3.02 2.97 0.106 0.108 2.55 2.49 0.098 0.100
2.19 2.14 0.085 0.087 1.92 1.86 0.081 0.084 1.51 1.45 0.088 0.091
1.31 1.26 0.093 0.097
1.312 1.431 1.521 1.621 1.685 1.847 1.910
Averages 0.094 0.097 zkO:OO8 ~kO.008
III. Rabbit skeletal myosin was found to have a light scattering
molecular weight of 550,000. The concentration dependence of the
scattering curve might also be interpreted as being indicative of
some type of interaction at higher protein concentrations.
Evidence of aggregation of cardiac myosin in the more con-
centrated solutions is seen in both the light scattering and dif-
fusion measurements. Contrariwise, the slope of the sedimenta- tion
plot de/& (Fig. 2) is relatively unaffected by concentration,
probably because it is a function of the cross section of a long
molecule. In fact if one plots D/s which is proportional to l/M
against HC/T (corrected for dissymmetry) which is also propor-
tional to l/M, good linearity is obtained over a range of
con-
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gave a value of 226,000 for dog heart myosin. The estimates from
equilibrium sedimentation and light scattering studies were,
respectively, 223,000 and 270,000. A total of nine preparations
were studied, with each preparation providing material for several
measurements so that interlocking curves were obtained over the
whole concentration range for each of the measurements.
The aim of achieving homogeneity of cardiac myosin in solu- tions
used for physicochemical measurements must not be compromised. The
presence of small amounts of impurities, such as a small leading or
trailing peak in the ultracentrifuge, should bc sufficient reason
to postpone physicochemical meas- urements until homogeneity is
obtained. Such impure prcpara- tions of cardiac myosin yield
falsely high molecular weights (5). In our ultracentrifugal
studies, leading peaks usually of actomyo- sin, always appeared in
the early precipitates even in the presence of liberal amounts of
ATP, and reprecipitations and preparative ultracentrifugations were
carried out until all fast componrnts were removed. In agreement
with Lowey and Holtzer’s observa- tions on the aggregation of
skeletal myosin (24), furthermore, we have noted that fast peaks
appear in ageing solutions of cardiac myosin: their presence
completely vitiates any valid physicochemical studies.
The molecular weight of cardiac myosin obtained in this study is
approximately one-half the currently accepted value for rabbit
skeletal myosin of 440,000 (16-19). Since the amino acid analyses
of cardiac myosin’ arc very similar to those of skeletal myosin,
dog heart cardiac and rabbit skeletal myosin may be related to each
other as monomer to dimer. Of further interest in this connection
is the finding of Kielley and Harrington (25) that under the
influence of guanidine salts, rabbit skeletal myosin may be
depolymerized to a monomer of 219,000 in molecular weight. Heavy
meromyosin, liberated from skeletal myosin by tryptic digestion
(14, 21, 26) has a lower molecular weight than skeletal myosin
estimated to range from 232,000 (27) to 324,000 (28). Although
H-meromyosin is closer to the sizo of cardiac myosin than its
parent molecule, it retains the full ATPase ac- tivity of skeletal
myosin. This fact suggests that subtle dif- ferences in protein
structure, apart from molecular weight, esist between cardiac and
skeletal myosin.
As regards the shape of cardiac myosin, the physical constants
previously described and the calculated molecular weight were
substituted into three equations for the determination of fric-
tional ratio (f/fO) (29). Essentially identical results were ob-
tained (2.14 to 2.15) as further evidence of the homogeneity of
these myosin preparations. Substitution of this value for f/f0 into
Perrin’s equation (30) gave an axial ratio for an ellipsoid of
revolution of 24: 1. Substitution of the limiting viscosity number
obtained for cardiac myosin in Simha’s shape equation (31) gave an
axial ratio of 23:l. The application of Scheraga and Mandelkern’s
formula (32, 33) for the calculation of “ef- fective hydrodynamic
volume” using so, DO, 0, for a single solvent at approximately the
same temperature (but without converting them to values at 20” in
water) gave B values of 2.77 X IO6 and 2.71 X lo6 from which axial
ratios of 29 and 27 were computed, in good agreement with the
estimates based on frictional ratio from viscosity alone.
From the axial ratio and the molecular weight, the dimensions of an
ellipsoidal cardiac myosin molecule would be 690 X 28 A. The
viscosity measurements rule out a sphere. From the molecular weight
and avrrage amino acid residue weight, a rigid
’ R. Iyengar et al., unpublished observations.
rod composed of a single a-helix (34) would be approximately 2,800
A x 10 A, a shape not consistent with the physical con- stants. A
fully random coil of this weight (35) would have a root-mean-square
end-to-end distance of 385 A.
The above considerations, of course, relate to the cardiac myosin
molecule at infinite dilution. The changes in viscosity, diffusion
constant, and HC/T with increasing concentration sug- gest that
end-to-end aggregation or possibly a coil to rod trans- formation
occurs in more concentrated solutions. Aggregation as a function of
concentration has been seen in studies of to- bacco mosaic virus
protein (36) and cy-casein (37). Even skeletal myosin has been
reported to show increased polydispcrsity with an increasing number
of shorter particles in dilute solution (38). What the state of
cardiac myosin is in the intact heart in which the concentration is
of t.he order of 8 g/100 g of tissue (2) and the ionic strength
much less than 0.6 is unknown. Our data are consistent with a
physiological model which possesses both some rigidity and some
random coil elasticity, which could provide the flrsibility
required in the contractile cycle.
SUMMARY
Cardiac myosin from normal dogs has been isolated and characterized
by physicochemical methods. From measure- ments of velocity
sedimentation, equilibrium sedimentation, diffusion, and partial
specific volume a molecular weight of 225,006 was obtained. Light
scattering measurements yielded a molecular weight of 270,000 and a
length for a coil model of 674 A. Jfeasurements of intrinsic
viscosity, in addition to the above measurements, were consistent
with a model for cardiac myosin of a coil 690 x 28 A. Aggregation
of these molecules appears to occur in concentrated solution. Since
normal cardiac myosin is different from any previously
characterized myosin, the name myosin C is proposed for it.
ilcknowledgment-The authors acknowledge the collaboration of
Doctors ?tI. L. Liang and Dorothy Piatnck in the preparation of the
animals for study and the expert technical assistance of hIr. L. E.
Wallen, Mrs. V. Bartlebaugh, and Miss D. Terrill.
RI’FERENCES
1. WEBER, H. H., AND PORTZEHL, H., in M. L. ANSON, K. BAILEY, AND
J. T. EDSALL (Editors), Advances in protein chemistry, Vol. VIZ,
Academic Press, Inc., New York, 1952, p. 161.
2. SZENT-GY~RGYI, A., Chemistry of muscular contraction, 2nd
edition, Academic Press, Inc., New York, 1951.
3. SZENT-GY~RQYI, A., Chemical physiology of contraction in body
and heart muscle, Academic Press, Inc., New York, 1953.
4. MOMMAERTS, W. F. H. M., Muscular contraction, Interscience
Publishers, New York, 1950.
__, .
8. CSAPO. A.. Ann. N. Y. Acad. Sci.. 76. 790 (1959). 6. OLSOH;
R.‘E., Am. J. Med., !ZO. li9 (i956).
10. ELLEKBO~EN, E., AKD OLSON, R. E., Federation Proc., 14, 207 (
1955)
11. STERN, H., ELLENBOGEN, E., ASD OLSON, R. E., Fe&ration
Proc., 16, 363 (1956).
12. RUPP, J. C., Ph.D. Thesis, Duke University, 1955. 13. ZIMM, B.
H., J. Chem. Phys., 16, 1093 (1948). 14. GERGELY, J., J. Biol.
Chem., 24IO. 543 (1953). 15. BALDWIN, R. L., Biochem. J., 66, 490
(1957). 16. VON HIPPEL, P. H., SCZHACHYAN, H. K., APPEL, P., AND
Mo-
RALES, 11. F., Biochim. et Hiophys. Acta, 28, 504 (1958).
by guest on A pril 14, 2019
http://w w
w .jbc.org/
D ow
nloaded from
2648 Characterization of Myosin from Normul Dog Heart Vol. 235, Ko.
9
17. MOMMAERTS, W. F. H. M., AND ALDRICH, B. B., Biochim. et
Biophys. Acta, 33, 627 (1958).
18. LAKI, K., AND CARROLL, W. R., Nature, (London), 176, 389
(1955).
19. HOLTZER, A., AND LOWEY, S., J. Am. Chem. Sot., 81. 1370
(1959).
20. DOTY, P., AND STEINER, R. F., J. Chem. Phys., 18, 1211 (1950).
21. GERGELY. J.. GOUVEA, M. A., AND KOHLER, H., Circulatia,
XIV, 940 (i956). 22. MI~LYI, E., AND SZENT-GY~RQYI, A. G., J. Biol.
Chem., 261,
189 (1953). 23. TENOR, Ml, AND SNELLMAN, O., B&him. et Biophys.
Acta,
16, 395 (1954). 24. LOWEY, S., AND HOLTZER, A., J. Am. Chem. Sot.,
81, 1378
(1959). 25. KIELLEY, W. W., AND HARRINQTON, W. F., Fe&ration
Proc.,
18, 259 (1959). 26. MIH~LYI, E., AND SZENT-GY~RGYI, A. G., J. Biol.
Chem., 291,
211 (1953). 27. SZENT-GY~ROYI, A. G., Arch. Biochem. Biophys., 42,
305
(1953).
28. LOWEY, S., AND HOLTZER, A., Biochim. et Biophys. Acta, 34, 470
(1959).
29. SVEDBRG,’ T., AND PEDERBEN, K. O., The ultracentrifuge, The
Clarendon Press, Oxford, 1940.
39. PERRIN, F., J. phys. radium (7), 7, 1 (1936). 31. SIMHA. R.. J.
Phus. Chem.. 44. 25 (19401 32. SCHER~GA; H. A.; AND M~ND~LKE&J,
21, J. Am. Chem. Sot.,
76. 179 (1953). 33. MANDELKERX, L., KRIGBAUM, W. R., SCHERAOA, H.
A., AND
FLORY, 1’. J., J. Chem. Phys., 30, 1392 (1952). 34. Low, B. W., in
H. NEURATH AND K. BAILEY (Editors), The
proteins, Vol. fA, Academic Press, Inc., New York, 1953, p.
235.
35. FLORY, P. J., AND Fox, T. G., JR., J. Polymer Sci., 6, 745
(1959).
36. LAUFFER, M. A., ANSEVIN, A. T., CARTWRIQHT, T. E., AND BRINTON,
C. C. JR., Nature, (London), 181, 1333 (1953).
37. VON HIPPEL, P. H., AND WAUQE, D. F., J. Am. Chem. Sot., 77.4311
(1955).
38. JOLY, M., SCHAPIRA, G., AND DREYFUB, J. C., Arch. B&hem.
Biophys., 69, 165 (1955).
by guest on A pril 14, 2019
http://w w
w .jbc.org/
D ow
nloaded from
1960, 235:2642-2648.J. Biol. Chem.
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