characterization of myosin from normal dog heart*
TRANSCRIPT
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.
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2648 Characterization of Myosin from Normul Dog Heart Vol. 235, Ko. 9
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1960, 235:2642-2648.J. Biol. Chem.
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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.
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