Characterization of an active, fluorescein-labelled kinesin.
Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125-kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF-kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF-kinesin binds to microtubules in the presence of adenosine 5'-[beta, gamma-imino]triphosphate or ADP. Brief incubation of the microtubule. AF-kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF-kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP.
Marya
P K
PK
Department of Biophysics, King's College London, England.
Fraylich
P E
PE
Eagles
P A
PA
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D000818:Animals; D002417:Cattle; D005452:Fluoresceins / Q000737:chemistry; D066298:In Vitro Techniques; D016547:Kinesins; D008870:Microtubules / Q000502:physiology*; D008970:Molecular Weight; D009068:Movement; D009419:Nerve Tissue Proteins / Q000302:isolation & purification
D005452:Fluoresceins; D009419:Nerve Tissue Proteins; C015296:5-iodoacetamidofluorescein; D000251:Adenosine Triphosphatases; D016547:Kinesins