Characterization of the microtubule movement produced by sea urchin egg kinesin.

M. E. Porter, J. M. Scholey, D. L. Stemple, G. P. Vigers, R. D. Vale, M. P. Sheetz, J. R. McIntosh

Research output: Contribution to journalArticle

85 Scopus citations

Abstract

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.

Original languageEnglish (US)
Pages (from-to)2794-2802
Number of pages9
JournalJournal of Biological Chemistry
Volume262
Issue number6
StatePublished - Feb 25 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Porter, M. E., Scholey, J. M., Stemple, D. L., Vigers, G. P., Vale, R. D., Sheetz, M. P., & McIntosh, J. R. (1987). Characterization of the microtubule movement produced by sea urchin egg kinesin. Journal of Biological Chemistry, 262(6), 2794-2802.