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 micrograms 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 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/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.
Urrutia
R
R
Laboratory of Cellular Biology, National Institute of Deafness and other Communication Disorders, Bethesda, Maryland 20892.
Kachar
B
B
D000313:Adrenal Medulla / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D016547:Kinesins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D007700:Kinetics; D008870:Microtubules / Q000201:enzymology; D013379:Substrate Specificity
D016547:Kinesins