Quantitative analysis of sea urchin egg kinesin-driven microtubule motility

S. A. Cohn, A. L. Ingold, J. M. Scholey

Research output: Contribution to journalArticlepeer-review

87 Scopus citations

Abstract

We have analyzed the effects of various substrates and inhibitors on the rates of microtubule (MT) motility induced by sea urchin egg kinesin using real-time computer analysis and video-enhanced light microscopy. In the presence of magnesium, 10 mM concentrations of all the nucleotides tested supported MT translocation, with velocities in MgATP > MgGTP > MgTTP ≃ MgUTP > MgCTP > MgITP. The velocity of kinesin-driven MT motility is fairly uniform over approximately 3 pH units, from pH 6 to 9, with almost no motility outside this range. In the presence of ATP, no motility is observed in the absence of divalent cations; addition of Mg2+ but not addition of Ca2+ restores motility. MgATP-dependent MT motility is reversibly inhibited by Mg-free ATP, EDTA, or tripolyphosphate, suggesting that Mg-free ATP is an inactive substrate analogue. MgATP and MgGTP both obey saturable, Michaelis-Menten kinetics, with apparent K(m) values of approximately 60 μM and 2 mM, and V(max) values of approximately 0.6 and 0.4 μm/s, respectively. MgATPγS and MgADP are classic competitive inhibitors of approximately 15 and 150 μM, respectively. Adenosine 5'-(β,γ-methylene)-triphosphate and N-ethylmaleimide only inhibit MT motility weakly, while adenyl-5'-yl imidodiphosphate and vanadate strongly inhibit MT motility, but not in a simple competitive manner. Moreover, in contrast to other inhibitors which cause a unimodal decrease in MT mean velocity, vanadate concentrations greater than ~10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.

Original languageEnglish (US)
Pages (from-to)4290-4297
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number8
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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