In vitro synthesis of native myosin, actin, and tropomyosin from embryonic chick polyribosomes.

There is a low level of free ribonuclease present in embryonic chick muscle, and the ribosomes are not attached to an endoplasmic reticulum. Thus, it is easy to isolate the polyribosomes (polysomes) in a relatively undegraded fashion. In a previous study we have shown that it is possible to use the undegraded polysomes isolated from embryonic chick leg muscle to form a highly active cell-free protein synthetic system.1 A class of large polysomes containing 50-60 ribosomes was shown to be active in the synthesis of myosin. In the present communication we identify the polysomes which carry out the synthesis of actin and tropomyosin-the other major fibrous proteins of muscle tissue. It is further shown that these proteins synthesized in vitro have the same chemical properties as those seen in native preparations. From the size of the polysomes and the size of the polypeptide chains, we conclude that all three of these fibrous proteins are being synthesized with monocistronic messenger RNA's. By observing changes in the polysomal pattern during different stages of embryonic development, it is inferred that actin-synthesizing polysomes are formed in large numbers initially, followed by myosin and, finally, tropomyosin polysomes at later stages of embryogenesis. Materials and Methods.-Methods for preparing the tissue homogenate from leg muscles of 14-day-old chick embryos were described previously.' Four polysome fractions, A-D, were isolated and pooled from six sucrose gradients. The polysomes from fractions A through D were sedimented and gently resuspended in buffer. Fraction A had 0.1 mg of ribosomes, B and C each had 0.2 mg, and D had 0.35 mg. An energy-generating system, radioactive amino acids, and a pH-5 enzyme fraction were added as described previously to make a total incubation volume of 1.0 ml for each of the four fractions.1 Incubation was carried out at 370C for 1 hr, 50 -y of ribonuclease was then added, and the incubation mixtures cooled to 00. The products of the incubation mixture were analyzed both by acrylamide gel electrophoresis' and by protein purification procedures. Myosin was prepared both from adult chicken pectoral muscle and from embryonic leg muscle.2 After incubation, mixtures A-D were each added to 4 ml of a solution containing 10 mg of purified myosin in KCl buffer: 0.5 M KCl, 0.01 M tris buffer (pH 7.4), and 0.001 M ethylenediaminetetraacetate (EDTA). The radioactive products of the cell-free incorporation were analyzed by carrying out a series of purification steps for isolating myosin at 3°C. At each step the radioactivity per milligram of protein was measured. An aliquot of protein was precipitated in 5% trichloroacetic acid and the precipitate collected on Millipore filters for radioactive counting. Protein content was determined by the method of Lowry." The specific activity of the initial mixture is plotted as step 1. The solution was diluted with water to lower the ionic strength to 0.03 M KCl to precipitate myosin. After centrifugation at 10,000 X g for 10 min, the supernatant was decanted and the precipitate redissolved in 0.5 M KCl buffer. Step 2 is the specific activity of the redissolved material. The ionic strength was then lowered to 0.28 M KCl to precipitate actomyosin. The solution was spun at 20,000 X g for 20 min, the supernatant decanted, and its specific activity is step 3. The ionic strength of the solution was next reduced to 0.03 by the addition of water and the precipitate was again centrifuged. After decanting, the precipitate was dissolved in 0.5 M KCl buffer for step 4. Saturated NH4SO4 was added to