Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide.
Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D016547:Kinesins; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D009419:Nerve Tissue Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins