The protocols described here have proved to be an effective method for preparation of kinesin suitable for biochemical, biophysical, and immunological analyses. Beginning with a 1.2-liter cytosolic extract of bovine brain containing ∼24 g of protein, 2 mg of ∼95% pure kinesin can be obtained within 2 days. There are four major enrichment steps, as summarized in Fig. 6 and Table I. Based on quantitative SDS-PAGE, we estimate that these steps result in a purification of more than 300-fold. The ATPase activity in the presence of microtubules is substantial, and the kinetic properties are consistent with cellular levels of ATP (Km ∼0.2 mM) and [figure presented] [table presented] microtubules (apparent Km for activation ∼1.9 μM) in the axon.9 Minor modifications should allow the procedure to be enlarged or reduced in scale, or adapted to the brains of other vertebrate species. The availability of such procedures will greatly facilitate future studies of the cell and molecular biology of kinesin.
ASJC Scopus subject areas
- Molecular Biology