CONTRACTILE PROTEIN FROM CRAYFISH TAIL MUSCLE

Abstract

Contractile protein (myosin B) was extracted and purified from tail muscle of the crayfish Cambarus clarkii. UV absorption spectrum of crayfish myosin B dissolved in 0.6 [image] KC1 showed a protein nature. The extinction coefficient ([epsilon]) 275 was 9 and [epsilon]275 / [epsilon]255 was 1.3. Acid-soluble purine-pentose content was 1.5 x 10-5 mole per g protein and nucleic acid purine-pentose content was 1 x 10-5 mole. Crayfish myosin B was quite soluble in 0.4 [image] KC1. Salting-out analysis with ammonium sulfate indicated the main component is actomyosin. Super-precipitation with ATP was clearly seen to take place in the range of 0.08-0.16 [image] KC1 concentrations. The phenomenon was most prominent in the presence of 0.1 [image] KC1 and 10-5 [image] MgCl2 at pH 7 at 25[degree]C.Ethylenediaminetetraacetate (EDTA) in concentrations higher than 10-4 [image] completely inhibited the precipitation. A drop of viscosity with ATP was observed in the presence of 0.6 [image] KC1. The ATP sensitivity of Weber and Portzehl was 100%. The recovery process was also observed to a considerable extent. ATP also caused a drop of turbidity of the myosin B solution, the maximal drop reaching 50%. The magnesium-activated apyrase activity and adenylate kinase activity were detected in the water-extract of crayfish muscle. No adenylate deaminase activity was demonstrated in crayfish myosin B. Crayfish myosin B had a true ATPase, activated by Ca. In the presence of 0.6 [image] KC1 and 10 m[image] Ca at 30[degree]C, the enzyme showed two pH optima, a higher one at 9 and a lower one at 6. In the presence of 0.6 [image] KC1 at pH 7-8, EDTA maximally enhanced the ATPase action, Qp being higher than 3000. The activating effect of Ca was sensitive to the inhibiting action of Mg. The Michaelis constant was 5 x 10-5 M in the presence of 10 m[image] Ca and 1 x 10-3 [image] in the presence of 10 m[image] EDTA. The ATPase action of crayfish myosin B was very sensitive to heavy metals, such as Cu or Zn. Para-chloromercuribenzoate (PCMB) easily blocked the enzyme action. However, PCMB in dilute concentrations rather enhanced the Ca-activated ATPase activity.