THE FREE-ENERGY FOR HYDROLYSIS OF A MICROTUBULE-BOUND NUCLEOTIDE TRIPHOSPHATE IS NEAR ZERO - ALL OF THE FREE-ENERGY FOR HYDROLYSIS IS STORED IN THE MICROTUBULE LATTICE

The standard free energy for hydrolysis of the GTP analogue guanylyl-( a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution , was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtu- bules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that th e majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described h ere were obtained from video microscopy measurements of the kinetics o f assembly and disassembly of GMPCPP-microtubules and GMPCP-microtubul es. It was possible to study GMPCPP-microtubules since GMPCPP is not h ydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassi um) Pipes buffer with glycerol, which reduced the halftime for GMPCPP hydrolysis from >10 h to similar to 10 min. The rate for tubulin-GMPCP P and tubulin-GMPCP subunit dissociation from microtubule ends were fo und to be about 0.65 and 128 s(-1), respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydro lysis.