Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes^1,2 and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles^3,4. Kinesin⁵, a microtubule-based motility protein which may participate in organelle transport and mitosis⁶, binds microtubules in a nucleo-tide-sensitive manner^5,7,8, and requires hydrolysable nucleotides to translocate microtubules over a glass surface ^9,10. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity^11,12 although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5′-adenylyl imido-diphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg²⁺ with EDTA, by addition of Na₃VO₄, or by addition of AMPPNP with or without Mg²⁺ Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg²⁺-ATPase activity.