Characterization of the Steady-State Mg²⁺-Dependent Adenosine 5′-Triphosphatase of Bovine Brain Kinesin in the Absence of Microtubules

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The kinetic properties of Mg²⁺-ATPase of bovine brain kinesin in the steady-state were studied in the absence of microtubules. A double-reciprocal plot of kinesin Mg²⁺-ATPase activity vs. ATP concentration indicated two distinct K_m values, 128 and 3.3μM. ATPase activity decreased with increase in pH from 6.0 to 7.0, increased as the pH was increased to 8.0, and then gradually decreased above pH 8.0. The rate of ATP hydrolysis was also affected by the KCl concentration. ATPase activity decreased with an increase in KCl concentration from 15 to 50mM, increased as the KCl concentration was increased further, and then decreased when KCl exceeded 200mM. From an Arrhenius plot of temperature dependency of Mg²⁺-ATPase activity, activation energy was estimated to be 25.3J/mol between 45 and 23°C and 87.9J/mol between 23 and 6°C. Both heavy and light chains in kinesin were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a 2.8-fold activation of Mg²⁺-ATPase in the absence of microtubules. These properties are more like smooth muscle myosin than skeletal muscle myosin.