Abstract
To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC.3.6.1.3) in situ, myosin was covalently bound to the surface of 2 kinds of films: collagen and Immunodyne™. On collagen films, it was bound either with 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide (EDC) or with dimethyl-3,3′-dithio bis(propionimidate) (DTP). The apparent K m for K −-ATP rose from 0.26 mM for free myosin in solution to 2–5 mM for covalently bound myosin, and maximum K −-ATPase activity was very low. With the other film, Immunodyne™ from Pall®, the maximum activity of bound myosin was 170 nmol per min per 1.5 cm 2 film. The apparent K m for K −-ATP was 2.1 mM when the incubation mixture was vigorously stirred, and the effect of stirring indicated that the kinetics of K −-ATP hydrolysis are limited by external diffusion. The large amount of myosin bound per unit of Immunodyne film surface permitted the study of Mg 2-ATPase activity, although it was 400–500 times less than the K −-ATPase activity. The apparently non-Michaelian kinetics of Mg 2-ATP hydrolysis are attributable to the external diffusion. The apparent Michaelis constant observed at low Mg 2−-ATP concentrations rose from 0.27 μM for myosin in solution to 5 μM for myosin bound to Immunodyne film.
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