Separation and Recombination of the Ethylene Glycol Bis(β-aminoethyl Ether)-N,N'-Tetraacetic Acid-sensitizing Factor Obtained from a Low Ionic Strength Extract of Natural Actomyosin

Abstract

Abstract The ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid-sensitizing factor extracted from natural actomyosin at low ionic strength was concentrated in the fraction obtained between 40 and 60% ammonium sulfate saturation. Fractionation within narrower salt limits and at low protein concentrations yielded a troponin-rich fraction at 40 to 53% saturation and a tropomyosin-rich fraction at 53 to 60% saturation. These ammonium sulfate fractions could be further purified by isoelectric precipitation at pH 4.6. The supernatant of the 40 to 53% fraction was shown to contain primarily troponin, while the precipitate of the 53 to 60% fraction was pure tropomyosin. It was found that for maximal EGTA-sensitizing factor activity a ratio of tropomyosin to troponin of 1:1.3, w/w, was required. This combination appears to form a complex which sediments as a single peak in the ultracentrifuge with a sedimentation constant of 5.15 S.