Purification of the relaxing protein of rabbit skeletal muscle.

It has been well accepted that in the presence of sufficient amounts of ATP and magnesium, the contractile protein of muscle, actomyosin or myosin B, exhibits a relaxation phenomenon when a small amount of calcium is removed from the system by "relaxing factor,"' or by calcium chelators such as 1,2-bis-(2-dicarboxymethylaminoethoxy) ethane (EGTA). Ebashi and his associates2 3 reported that this relaxation phenomenon occurs only in the presence of a newly discovered protein in the contractile protein preparation. This new protein was called by Ebashi "tropomyosin-like protein," "native tropomyosin," or "troponin." However, we shall call it by a single term, "relaxing protein." Ebashi and Ebashi3 obtained a "partially purified preparation" and a "purified preparation" of the relaxing protein, but they reported that the "purified preparation" was chemically more pure but weaker in relaxing activity than the "partially purified preparation." Using Szent-Gyorgyi and Kaminer's metin preparations as a starting material, Azuma and Watanabe5' 6 showed that a fraction precipitated at 50 per cent saturation with ammonium sulfate was active as a relaxing protein. We have therefore applied salting-out with ammonium sulfate for purification of Ebashi and Ebashi's relaxing protein, and have been able to obtain a preparation chromatographically as pure as their "purified preparation" and as active in relaxing activity as their "partially purified preparation." Moreover, gel filtration achieved a further purification of the relaxing protein. Materials and Methods.-Rabbit back and leg muscles were used for preparing relaxing protein and myosin B. "Crude extract without acetone treatment," "partially purified preparation," and "purified preparation" of relaxing protein were prepared according to Ebashi and Ebashi.3 Trypsin-treated myosin B was prepared according to Maruyama et al.,7 but the myosin B suspension (1 mg protein per ml of 0.1 Al KCl buffered with 20 mM Tris-maleate, pH 7) was incubated with trypsin (10 ,ug per ml) for 10 min at 250C instead of 30 min at 20'C. For chromatographic fractionation of various proteins, a column bed 2.5 cm in diameter, 85-95 cm in height, and containing approximately 15 gm (dry wt) of Sephadex G-200 was used. The chromatographic medium was 0.3 M KCl buffered with 20 mM Tris-HCl (pH 7.5), and the flow rate was 15-40 ml per hr. The operating temperature was about 50C. The collecting tubes contained 5-10 ml each, and the absorbance at 278 myA (sometimes also at 260 m~i) was measured for each tube. ATPase activity of the trypsin-treated myosin B was measured by determining orthophosphate liberated from ATP. Superprecipitation was measured by the increase in absorbance at 550 mju upon addition of ATP.8 Protein was determined by the Biuret reaction,9 using bovine serum albumin (Sigma Chemical Co.) as a standard. One milligram per milliliter albumin (6.25 X nitrogen) gave an absorbance of 0.064 at 560 mju in the Biuret reaction. Tryptophan content, ribose content, and