Myosin was prepared from the extensor digitorum longus, gastrocnemius medialis and lateralis, tibialis anterior, flexor of the claws of the anterior limb, and diaphragm muscles of the cat and didactyl sloth (Choloepus hoffmanni Peters). Actin-activated, Ca2f-activated, and EDTA-activated ATPase activities of the myosins from cat muscles were two to four times higher than those of the myosins from the same muscles of the sloth. The difference in the Ca2+-activated ATPase activity was found in the pH range of 5.5 to 10.0, and in the KCl concentration range of 50 mM to 500 mM a t pH 7.0.The kinetic Characteristics of the two myosins indicated that both the K , and the Vmax of the ATPase activities of the sloth myosin were lower than those of cat myosin by a factor of several-fold.Reconstituted cat actomyosin superprecipitated four to six times faster than sloth actomyosin. This difference in the superprecipitation did not vary appreciably when the concentration of ATP and the ratio of myosin to actin were varied. A correlation between the ATPase activity and the speed of superprecipitation of actomyosins of the cat and sloth was shown.The contraction time of various muscles of the cat and sloth was found to be inversely proportional to the actin-activated ATPase activity of their respective myosins. The speed constant of shortening of the muscles of the diaphragm appeared to be proportional to the actin-activated ATPase activity of their myosin.I n contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the actin-binding ability of myosins of the cat and sloth was rather constant. The extent of superprecipitation of various reconstituted actomyosins of the cat and sloth was also the same. These properties of the isolated myosins were related to the similar tension output of the muscles of the cat and sloth.
Merocyanine binds extensively to rat liver mitochondria in spite of the presence of a sulfonic acid group which would suggest only limited penetration through the membrane. Passive binding shows both tight and weak binding components and is dependent on salt concentration and ionic strength in accord with the Gouy-Chapman theory. The binding of merocyanine to mitochondria is accompanied by both a fluorescence enhancement and a spectral shift. Induction of an electrical field by either respiration or K+ diffusion potential results in a partial reversal of the spectral shift seen on dye binding. At low temperature, the merocyanine spectral response to an electrical field is biphasic, consisting of a fast phase with a t1/2 of less than 1 sec at 15 degrees C and a slower phase which may vary considerably in rate and extent with conditions. The spectral shift during the two phases appears similar, but differ in sensitivity to ionic strength and temperature. The spectral shift during the fast phase at 15 degrees C indicates that the major component is a decrease in bound monomer and an increase in the aqueous dimer, indicating an "on-off" mechanism. It is suggested that the fast and slow phases of the merocyanine response may be due to two different populations of dye, possibly located at the outer and inner surfaces, respectively, of the mitochondrial membrane. The electrophoretic movement of the dye located in the membrane interior would result in the temperature-sensitive slow phase response. Demonstration of the proportionality of the fast phase response to the magnitude of the membrane potential suggests the usefulness of merocyanine in studies with mitochondrial systems.
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