A Protein Factor Regulating the State of Aggregationof Myosin*

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Abstract
A regulatory factor, which changes dramatically and reversibly the state of aggregation of myosin, was found in the metin preparation of Szent-Györgyi and Kaminer. The factor, named “myosin aggregation factor” (MAF), was inactivated at 60°, and precipitated at pH 5.0. It was soluble in 0.1M K.C1 at pH 7.0, was salted out from 20 to 55 per cent saturated ammonium sulfate, and was precipitated in O.lM KC1 by ultracentrifugation at 35,000 r.p.m. for 3 hours. When the solution from the precipitate was dialyzed against an ATP solution, it showed the myosin aggregation activity only slightly, even after the addition of 0.1M KC1 and 2mM MgCl2. Its ultraviolet absorption spectrum exhibited a peak at 280 mμi. The factor lost its activity by digestion with Pronase-P, but not with RNase-A [EC 2.7.7.16], indicating that the factor is a protein. Myosin and MAF formed a complex, which in the absence of ATP showed spherical aggregates in 0.1M KC1 and 2mM MgCl2, at pH 7.0. These aggregates were transformed into a net-work of filaments upon addition of ATP. After splitting the ATP, the complex formed again the spherical aggregates. Magnesium ion and a minute amount of calcium ion had to be present in order to induce the above transition. By measuring the effect of the supernatant obtained by centrifuging a mixture of myosin and MAF preparation on the aggregation state of myosin and its protein content, it was concluded that the amount of MAF binding with myosin is about 10 per cent by weight of myosin, while its aggregation effect is already saturated by the addition of only about 1 per cent MAF by weight of myosin. H-Meromyosin in O.lMKCl was insolubilized by the addition of MAF. The effect was saturated by the addition of 4 per cent MAF by weight of H-meromyosin. The complex of H-meromyosin and MAF was solu-bilized on the addition of ATP, and became insoluble again after ATP was split.

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