Isolation of the microtubule-vesicle motor kinesin from rat liver: selective inhibition by cholestatic bile acids.
OBJECTIVE
Vesicular transport is supported by microtubule-based, force-transducing adenosine triphosphatases (ATPases), such as kinesin, a ubiquitous motor enzyme that has been well studied in neuronal tissues. Although vesicular transport is important for hepatocellular secretory and clearance activities, the role of kinesin in liver function is poorly understood. Furthermore, the effects of bile acids on kinesin are unknown.


METHODS
Kinesin was purified from rat liver cytosol by conventional chromatography and microtubule affinity binding and was characterized by immunoblotting with domain-specific kinesin antibodies and amino acid sequencing of tryptic fragments. Kinesin activity was measured with and without bile acids using an in vitro motility assay and ATPase assays.


RESULTS
Immunoblot analysis and partial amino acid sequencing of purified kinesin showed that the sequence at the heavy chain of hepatic kinesin is nearly identical to that of brain kinesin. Purified kinesin transported microtubules in vitro with a velocity of approximately 0.5 microns/s; this activity was significantly inhibited by 0.5-1 mmol/L taurochenodeoxycholate but not by tauroursodeoxycholate. At a dose of 1 mmol/L, chenodeoxycholate conjugates, but not ursodeoxycholate or cholate conjugates, directly inhibited the ATPase activities of kinesin and another microtubule motor, cytoplasmic dynein.


CONCLUSIONS
Cholestatic concentrations of chenodeoxycholate conjugates directly inhibit the activity of microtubule motors, suggesting a possible mechanism for impairment of vesicular transport in cholestasis.
Marks
D L
DL
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota.
LaRusso
N F
NF
McNiven
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002635:Chenodeoxycholic Acid / Q000494:pharmacology; D002779:Cholestasis / Q000378:metabolism*; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification*; D008099:Liver / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology
D001647:Bile Acids and Salts; D002635:Chenodeoxycholic Acid; D013655:Taurochenodeoxycholic Acid; D016547:Kinesins