Biochemical Characterization of ATPase Activity of Dynamitin / 中国生物化学与分子生物学报

ABSTRACT

Dynactin is a multi-subunit complex that has been implicated in the function of cytoplasmic dynein which is a minus end - directed microtubule motor protein with numerous functions including nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during interphase and mitosis. Dynamitin,the 50 kD subunit of dynactin, is important for stabilizing the dynactin complex. To gain more insight into the mechanism of stabilizing, we analyzed the sequence of dynamitin and revealed that domains of dynamitin is homology to the Walker A and Walker B ATPase motifs. The purified GST-dynamitin and GST-free dynamitin both showed ATPase activity specifically. An inactivating mutation in the Walker A, but not the Walker B ATPase motif abolished the ATPase activity of dynamitin. The mutational analysis studies further supported that dynamitin is an ATPase. Kinetic studies of the ATPase activity of dynamitin revealed a Km for ATP of 125.78μmol/L and a kcat of 7.4 min-1.