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Miller, E. J., Lane. J. M., and Piez, K . A. (1969), Biochemistry 8, 30. Miller, E. J.. Martin, G. R . , Piez, K . A,, and Powers. M. J . (1967). J. Bioi. Clieni. 242, 5481. Miller, E. J., and Piez, K. A. (1966), A n d . Biochem. Ih, 320. Nagai. Y . ,Gross. J . , and Piez, K . A . (1964), Ann. N . 1’. Acud. Sci. 121, 494. Paz, M. A., Blumenfeld, 0. O., Rojkind. M., Henson, E., Furfine. C., and Gallop. P. M. (1965). Arch. Biochern. Biophys. 109, 548. Paz. M. A., Lent, R. W., Faris, B., Franzblau, C., Blumenfeld, 0. O., and Gallop, P. M. (1969), Biochem. Biophys. Res.
Commun. 34, 221. Piez, K . A,, Bladen, H. A., Bornstein, P., Butler, W. T., Kang, A. H., Lane, J. M., and Miller, E. J. (1969), Brookliacen S p n p . Biol. 21, 345. Piez, K. A,, Eigner, E. A,. and Lewis. M . S . (1963) Biochernistrj 2, 58. Piez, K. A , , Martin. G . R., Kang, A. H., and Bornstein, I-’. (1966), Bioc/ieniistrj, 5, 3813. Piez, K. A,, Weiss, E., and Lewis, M. S. (1960), J . Bioi. Chem. 235,1987. Pinnell, S . R., and Martin, G. R. (1968), Proc. Nut/. Acad. Sci. U. S . 61, 708.
Characterization of Rat Heart Myosin. I. Isolation and Physical Properties” R. L. McCarl and S. S. Margossiant
Cardiac myosin from rats was isolated and characterized by physical methods. The myosin appeared homogeneous by sedimentation velocity and sedimentation equilibrium centrifugation. The calculated value for was 6.35 S, and D ~ o , obtained , by the porous disk method was
ABSTRACT:
E
xtensive experimental data are now available concerning both the enzymatic and structural properties of rabbit skeletal myosin. Brahms and Kay (1963) reported certain variations in enzymatic properties between dog cardiac and rabbit skeletal myosins. On the other hand, Mueller et al. (1964) did not report any drastic differences between dog cardiac, dog skeletal, and rabbit skeletal myosins. The observed variations were ascribed to differences due to species and not due to inherent variations in the size and shape of the myosin molecule from different tissues. The observation (McCarl et a/., 1965) that cortisol acetate had a positive inotropic effect on rat heart cells in culture led us to isolate and purify myosin from mature rat cardiac tissue. In this present paper, a detailed analysis of the hydrodynamic properties of rat cardiac myosin will be presented. Experimental Procedures Materials ATP, the disodium salt, was obtained from Calbiochem, Los Angeles, Calif.
* From the Department of Biochemistry, College of Science, The Pennsylvania State University, University Park, Pennsylvania 16802. ReceicedJanuarj> 2, 1969. The authors thank Dr. S . Lowey of Children’s Cancer Research Fcundation, Bcston, Mass., for her criticisms in the preparation of this manuscript. t Present address’ Children’s Cancer Research Fcundation, Inc., Boston, Mass. 021 15.
1.56 X lo-’ cm2/sec. The apparent molecular weight from sedimentation equilibrium runs was of the order of 436,000. Conformational studies gave a value of 53 for the helical content of myosin isolated from rat cardiac tissue.
The animals used throughout these experiments were mature albino rats (obtained from the small animal laboratory in Frear Laboratories zt The Pennsylvania State University). In each isolation the hearts from 30 animals were pooled and extracted. The weight of rat cardiac tissue in each experiment was 37 ?E 3 g. Method3 Isolation of Rat Cardiac Mjwsin. The rats were killed by a blow on the head and the hearts were immediately excised and chilled. Blood was washed away with cold, deionized water and the hearts were minced. The minced tissue was homogenized in a Virtis homogenizer for 30 sec at half-maximal speed, at 0’. The homogenization was performed in three volumes of extracting solvent which consisted of 0.4 M KCI0.075 M KH2P04-0.075 M K-HP04-0.002 M ATP (pH 6.7). Following homogenization the extraction of myosin was continued for 10 min with constant stirring. Our method of isolating myosin was that described by Mueller (1964) except for a final purification by chromatography on a DEAE-cellulose column equilibrated with 0.2 M KCl-0.01 M Tris (pH 7.6) {Brahms, 1959). Elution from the DEAE-cellulose column was performed by applying an ascending KCI gradient (0.21.0 M KC1). The protein concentration in each 10-ml effluent fraction was then determined. The flow rate was regulated to 0.7 ml/min. Protein Determination. Protein was determined by a modification of Lowry’s method as described by Oyama and Eagle
PHYSICAL CHARACTERISTICS OF RAT CARDIAC MYOSIN
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(1956). On later samples, protein was determined by the spectr6hotometric method of Groves et al. (1968). This latter method was used in view of the fact that the agreement between the two methods was very good and the latter took less 1.2 time. Analytical Ultracentri/ugarion. SEDIMENTATION COEFFI1.0 CIENT. The sedimentation coefficient of rat cardiac myosin was determined by following the migration of the boundary of the protein peak by an ultraviolet-scanning system. A Kel-F double-sector centerpiece was used with one sector filled with the buffer and the other with the protein solution. The runs were made at 60,000 rpm and 20". Both the function pen and the derivative pen were employed 0.2 , in these experiments. A scan speed of 3.02 cmlmin and a chart speed of either 1 or 5 mm per sec were used. The sedimentation 0.0 ' 0.2 coefficient of rat cardiac myosin was determined at three difI 4 8 12 16 2 0 2 4 2 8 32 36 ferent protein concentrations and extrapolated to infinite diFRACTION NUMBER lution. MOLECULAR WEIGHT.The ultraviolet-scanning system of FIGURE 1 : Chromatography of myosin on a 14 X 2.5 cm column of DEAE-cellulose. A gradient elution of 0.2 M KCI-O.01 M Tris (pH the analytical centrifuge was used to calculate the molecular 7.6) to 1.0 M KCI-0.01 M Tris (pH 7.6) was used. Flow rate was 0.7 weight of rat cardiac myosin. In these experiments, one of the mllmin and IGml fractions were collected. compartments of the Kel-F double-sector centerpiece had the protein solution layered on FC-43, and the other compartment was filled with the buffer. The column height in different runs ranged from 2.0 to 2.2 mm. At the start of centrifugation, the rotor was accelerated to 18,OOO rpm for 3 hr after which time it was decelerated to6377 rpm and kept at that speed until equilibrium was reached within 27-30 hr. The molecular weight was calculated according to M,, = ZRT/(I - Vp)w,)wydIn c/dr'), where R, T, P, and w have the usual meaning and a value of 0.728 ml/mg was used for V (Schachman, 1959); d In c/drZis the concentration gradient obtained from the slope of the plot I 4 I 20 I 28 I of In c us. rs. MINUTES As a control, rabbit skeletal myosin was isolated and purified in a similar fashion and its molecular weight was also FIGURE 2: Sedimentation velocity patterns of rat cardiac myosin using an ultraviolet scanning system. Upper curve: e us. r; lower determined as described above. curve: dridx os. 1. Protein concentration: 1.00 rnglrnl; speed: 60,oOO Diffusion Coesfcients. The diffusion coefficient was calcurpm ai 20". The time intervals in minutes at which the Scans were lated by the porous disk method as described by Northrop taken are 4, 20, and 28, respectively, reading from left to right. et al. (1948). The experiments were performed in a constant temperature water bath at 20 f O.O0lo. The diffusion cell was standardized by using 2 M NaCl solution (Anson and Northrop, 1937). The amount of NaCl at tubes 14 and 15 which did not possess any activity. The diffused at various intervals was determined by titration with A2no/A2soratio of the major peak was greater than 1.70 indi0.101 M AgNOa. The cell constant was then calculated accordcating no contamination with nucleic acids as reported elseing to K = Dt Cx/C,V,, where K is the cell constant; D = difwhere (Gaetjens et a/., 1968). fusion constant of 2 M NaCl at 20'; r is the time in seconds of Figure 2 is a photographic representation of ultraviolet diffusion; CIand CZ,concentrations of NaCl inside and outside scans of a sedimentation velocity run in the analytical ultrathe cell, respectively; V, is the volume outside the cell. The centrifuge. In each picture, the upper and lower curves repdiffusion coefficient of myosin could then be estimated accordresent concentration, e, us. distance from the axis of rotation, ing to D = 2.3KV1V2/(V, Vdr . log v 8 - ( V , V,)Q./ r, and dc/dr us. r. respectively. The derivative curve reveals a V,S - (VI V J Q , where V, is the volume inside the cell; S, single peak and no anomaly can be detected in the boundary the total solute present; Q., quantity of solute outside the cell patterns of the integral curve. Neither of these curves reveal at t = 0; and Q, quantity of solute diffused at time, f . any high molecular weight contaminant in the preparations. The diffusion coefficient was determined at three different Furthermore, the plot of In c us. r z (Figure 3) is straight and concentrations of proteins and extrapolated to zero concendoes not suggest polydispersity. tration. Sedimentation Coesfcient. The calculated values of smlX at three different myosin concentrations showed a concentration dependence similar t o that reported for myosin isolated from Results other sources (Stracher and Dreizen, 1966) with a sedimentaHumogeneity of Preparations. Figure 1 shows the results of tion coefficient at infinite dilution, &,,-, of 6.35 S (Figure 4). chromatography on DEAE-cellulose column. A single peak It should be pointed out that sedimentation coefficient calwith ATPaseactivity wasobtained,followed by a smallshoulder culations were made on fresh myosin preparations within 4 hr
I'
0.4~,;1-,;~~,,~
+
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MCCARL AND MARGOSSIAN
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19:69 the difusion coefficient of rat cardiac myosin was estimated at three different myosin concentrations and the values obtained for Dzo,\,were plotted against concentration (Figure 5). From this figure it can be seen that the concentration dependence of the diffusion coefficient is similar to that of rabbit skeletal myosin (Parrish and Mommaerts, 1954). Upon extrapolating the linear plot t o zero concentration, a value of 1.56 X lo-' cm2/sec was obtained for Molecular Weight. Although the In c cs. r 2 plot in Figure 3 suggests a monodisperse prepararion, 0.05 M phosphate was included in the solvent to prevent possible aggregation (Lowey and Holtzer, 1959). However, aggregation of myosin could still be detected on prolonged centrifugation (30 hr). Table I summarizes the molecular weights determined by
,
47
48
49
50
TABLE I : Summary of the Molecular Weight Determinations of Rat Cardiac and Rabbit Skeletal Myosin.cL
r2
3: Sedimentation equilibrium run of rat cardiac myosin. A representative plot of In C cs. r z . Protein concentration, 0.75 mg/ml. Equilibrium speed, 6377 rpm at 4".
FIGURE
Concn Source (mgiml) Rat Cardiac
I -
z i
m"
Prepn I
Prepn I1
0.67
432,000
435,000
1 23 2.30
432,000 446,000
433,000 448,000
Av
5 I-
Rabbit Skeletal
I
'\
~
1 ,oo
438,000
+ 8,100
466,000 - ._____-
For details of experimental techniques, refer to the section ---2 on Methods in the text. 1.00 1.25 if
,
4L-_--
436,000 d= 8,100
0.50
rng PROTEIN/ m l
Concentration dependence of the sedimentation coetIicient of rat cardiac myosin at 60,000 rpm and at 20". Values are extrapolated to infinite dilution.
FIGURE 4:
sedimentation equilibrium. Averages of duplicate runs on two different preparations are shown at three different concentrations of protein. In view of the fact that the range of protein concentration used is rather narrow, a plot l/M,,,,,,TS. concentration was not feasible. In order to calculate the molecular weight at infinite dilution, however, L I S ~is made of the relation 1/MilPr,= l/M; 2BC, where B is the second virial coefficient and C is concentration. If a value of 0.80 x 10-4 mole ml/g2 (Mueller et al., 1964) for B is assumed, the calculated value for M ; is found to be 485,000 which is in the same range of values reported for rabbit skeletal myosin. The sedimentation equilibrium molecular weight of rabbit skeletal myosin (used as a control in these experiments) was 517,000, applying a similar correction for nonideality. This value compares favorably with a molecular weight of around 500,000 generally accepted for rabbit skeletal myosin (Stracher and Dreizen, 1966). It should be noted that the molecular weight calculated from s:,), and D:",,in the Svedberg equation (415,000) is lower than the absolute value found from equilibrium sedimentation measurements. The low molecular weight is probably a reflection of experimental error in the diffusion coefficient. Diffusion coefficients reported for myosin are usually about 1.1 x cm2/sec(Stracher and Dreizen, 1966); the higher value is most likely due to limitations in the porous disk method
+
I
0
'/
c.5
;.50
1.30
2.30
rng PROTEIN/ m i
5 : Concentration dependence of the diffusion coefficient of rat cardiac myosin at 20". Values are extrapolated to infinite dilution.
FIGURE
after the samples were eluted from the DEAE-cellulose column. Difision CoefJicient. In order to determine the diffusion coefficient by the porous disk method, it was necessary to estimate the cell constant K . The calculated value for the cell used in these experiments was found to be 0.418 cm-l. Subsequently,
P H Y S I C A L
C H A I < A C I E R I S ~ I C S0 1 K A T
C'AKDIAC
M Y O S I N
3657
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FIGURE6: Moffitt plot of rat cardiac and rabbit skeletal myosin. The calculated value of bo for rat cardiac myosin is -335 while that of rabbit skeletal myosin is -379. (-0-0-) Rat cardiac myosin. (-e-@--) Rabbit skeletal myosin.
rather than a reflection of any change in the shape and hydration of the rat heart myosin. Helicity. So far the discussions have centered around the physical properties of rat cardiac myosin. In order to have a general idea of its conformation, namely its secondary structure, the helical content of this protein was estimated from optical rotatory dispersion studies. From the Moffitt-Yang plot in Figure 6, the calculated bo for rat cardiac myosin was found to be -334 (h) corresponding to a helical content of 53%. This value is lower than that of dog cardiac myosin which was reported to be 58 % (Kay et ai., 1964). A sample of rabbit skeletal myosin was also prepared and the calculated value for the helical content was 60 which is well within the range of 60-65x reported by Mommaerts (1966) from circular dichroism studies. Discussion In the present study, the method used to isolate cardiac myosin was essentially that of Szent-Gyorgyi (1951) except for additional chromatography on DEAE-cellulose to free myosin from nucleic acid contaminations. It should be pointed out that rat cardiac myosin vias not resolved into two discrete peaks on chromatography as was reported in the case of rabbit skeletal muscle myosin (Brahms, 1959). In their studies of dog cardiac myosin, Brahms and Kay (1962) reported a molecular weight of 750,000. This value is significantly higher than the value usually reported for myosin. In a separate investigation, Mueller et al. (1964) using a"mu1tispeed" Archibald method obtained a value of 526,000 with a mole ml/g2. In the second virial coefficient of 0.80 X present investigation, the apparent molecular weight of rat cardiac myosin is of the order of 436,000 which is in the correct range. In these experiments, the final yield of myosin was rather low due to a high actomyosin content in the original extrac-
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tion, and also due to relatively little starting material. Hence, all these factors did not permit us to perform an adequate extrapolation to calculate the molecular weight at infinite dilution. If, however, one assumes the virial coefficient reported for canine cardiac myosin (Mueller er ui., 1964), the calculated molecular weight for rat cardiac myosin becomes comparable to rabbit skeletal myosin. Similarly, the sedimentation coefficient of rat cardiac myosin compares well with the values reported for skeletal muscle myosin. It was pointed out earlier that D:(,,wfor rat cardiac myosin differed from that of either rabbit skeletal muscle or canine cardiac myosin. This difference may be ascribed to a possible inadequacy of the porous disk used in these experiments for the free diffusion of a fibrous protein. In conclusion, then, the calculated values of molecular weight and sedimentation coefficients of rat cardiac myosin suggest that this protein is similar in size and shape to myosin isolated from rabbit skeletal muscle and dog skeletal and cardiac muscles. References Anson, M. L., and Northrop, J. H. (1937), J . Gen. Physiol. 17,393. Brahms, J. (1959), J. Am. Chem. Soc. 81,4997. Brahms, J., and Kay, C. M. (1962), J . Mol. B i d . 5, 132. Brahms, J., and Kay, C. M. (1963), J . Biol. Chem. 238, 198. Gaetjens, E., Barany, K., Bailin, G . , Oppenheimer, H., and Barany, M. (1968), Arch. Biochem. Biophys. 123,82. Groves, W. E., Davis, F. C. Jr., and Sells, B. H. (1968), Anal. Biochem. 22, 195. Kay, C. M., Green, W. A., and Oikawa, K. (1964), Arch. Biochem. Biophys. 108,89. Lowey, S., and Holtzer, A. (1959), J . Am. Chem. Soc. 81, 1378. McCarl, R . L., Szuhaj, B., and Houlihan, R. (1965), Science 150,1611. Mommaerts, W. F. H. M. (1966), J . Mol. Bioi. 15, 377. Mueller, H. (1964), J . Biol. Chem. 239,797. Mueller, H., Franzen J., Rice, R. V., and Olson, R . E. (1964), J . Bioi. Chem. 239, 1447. Northrop, J. H., Kunitz, M., and Herriott, R. M. (1948), Crystalline Enzymes, New York, N. Y . , Columbia University. Oyama, V. I., and Eagle, H . (1956), Proc. Soc. Exprl. Biol. Med. 91, 305. Parrish, R. G., and Mommaerts, W. F. H . M. (1954), J . Biol. Chem. 209,901. Schachman, H . K. (1959), Ultracentrifugation in Biochemistry, New York, N. Y., Academic. Stracher, A., and Dreizen, P. (1966), in Current Topics in Bioenergetics, Vol. 1, Sanadi, D . R., Ed., New York, N. Y.. Academic. Szent-Gyorgyi, A. (1951), Chemistry of Muscular Contraction, 2nd ed, New York, N. Y., Academic.
