ISOLATION OF THE MICROTUBULE-VESICLE MOTOR KINESIN FROM RAT-LIVER - SELECTIVE-INHIBITION BY CHOLESTATIC BILE-ACIDS

Background/Aims: Vesicular transport is supported by microtubule-based , force-transducing adenosine triphosphatases (ATPases), such as kines in, a ubiquitous motor enzyme that has been well studied in neuronal t issues. Although vesicular transport is important for hepatocellular s ecretory and clearance activities, the role of kinesin in liver functi on is poorly understood. Furthermore, the effects of bile acids on kin esin are unknown. Methods: Kinesin was purified from rat liver cytosol by conventional chromatography and microtubule affinity binding and w as characterized by immunoblotting with domain-specific kinesin antibo dies and amino acid sequencing of tryptic fragments. Kinesin activity was measured with and without bile acids using an in vitro motility as say and ATPase assays. Results: Immunoblot analysis and partial amino acid sequencing of purified kinesin showed that the sequence at the he avy chain of hepatic kinesin is nearly identical to that of brain kine sin. Purified kinesin transported microtubules in vitro with a velocit y of similar to 0.5 mu m/s; this activity was significantly inhibited by 0.5-1 mmol/L taurochenodeoxycholate but not by tauroursodeoxycholat e. At a dose of 1 mmol/L, chenodeoxycholate conjugates, but not ursode oxycholate or cholate conjugates, directly inhibited the ATPase activi ties of kinesin and another microtubule motor, cytoplasmic dynein. Con clusions: Cholestatic concentrations of chenodeoxycholate conjugates d irectly inhibit the activity of microtubule motors, suggesting a possi ble mechanism for impairment of vesicular transport in cholestasis.