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 greater than MgGTP greater than MgTTP approximately equal to MgUTP greater than MgCTP greater than 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 Km values of approximately 60 microM and 2 mM, and Vmax values of approximately 0.6 and 0.4 microns/s, respectively. MgATP gamma S and MgADP are classic competitive inhibitors of kinesin-driven motility in MgATP, with Ki values of approximately 15 and 150 microM, respectively. Adenosine 5'-(beta, gamma-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 approximately 10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.
Highlights
Addition of AMPPNP, Vi, or AMPPCP inhibited MT gliding, none of these inhibitors displayed competitive inhibition kinetics
Only MgGTP appears capable of supporting kinesindriven translocation at velocities >50% of that caused by MgATP, kinesin's broad range of nucleotide specificity allows cells the possibility of maintaining somelevel of kinesin activity under a wider range of conditions than the dyneinMT system
We find that nucleotides in the absence of M F cannot support motility
Summary
Addition of AMPPNP, Vi, or AMPPCP inhibited MT gliding, none of these inhibitors displayed competitive inhibition kinetics. Vi was the most potent of these inhibitors, causing complete inactivation of motility when added in concentrations about 10%that of MgATP (Fig. 8 in the Miniprint). AMPPNP inhibited MT gliding with kinetics that wereclose to competitive at low AMPPNP concentrations, butdisplayeddeviations from competitive inhibition kinetics asitsconcentrations were increased (Fig. 10 in the Miniprint). The effects of AMPPNP and Vi did not seem to be affected by the presence or absence of Mg+. AMPPCP was the weakest of the inhibitors tested, requiring concentrations in great excess over MgATP before significant inhibition occurred (Fig. 11 in theMiniprint). Much of AMPPCP’s effectiveness is reduced in the presence of equimolar Mg+.
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