An improved method for the purification of kinesin from bovine adrenal medulla

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 μg of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca⁺⁺ATPase of 0.4 μmol/min per mg and a Mg⁺⁺ATPase of 0.03 μmol/min per mg in the absence of microtubules. The addition of microtubules (5 μM) activates the Mg⁺⁺ATPase activity by almost 70-fold to a value of 1.9 μmol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.