| Abstract: | Kinesin is a mechano-chemical ATPase capable to move particles along microtubules and microtubules along the solid substrate. Molecule of bovine brain kinesin is a heterotetrameric unit consisting of two heavy (120 kDa) and two light (62 kDa) chains. We used limited proteolysis to study the location of the functional sites on the kinesin molecule. Chymotrypsin cleavage produced a stable 45 kDa fragment of the heavy chain which was purified from the digest using FPLC chromatography on a Superose 12 column. 45 kDa fragment contained both a microtubule-binding site and a ATPase site of the kinesin molecule. Cleavage of the 45 kDa fragment from the rest of the heavy chain significantly activated its ATPase activity. However, this activity remained fully dependent on microtubules. We suggest that the chymotrypsin cleavage uncouple ATPase activity of kinesin (found in the 45 kDa fragment) from its translocator activity (which, probably, required the presence of other parts of the molecule). |
