GTP hydrolysis during microtubule assembly.
The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.
D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D005990:Glycerol / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000032:analysis; D006160:Guanosine Triphosphate / Q000502:physiology*; D006868:Hydrolysis; D008722:Methods; D008870:Microtubules / Q000378:metabolism*; D008982:Molybdenum; D010756:Phosphoric Acids; D010761:Phosphorus Radioisotopes; D013552:Swine; D013997:Time Factors; D014404:Tubulin / Q000302:isolation & purification
D002021:Buffers; D010756:Phosphoric Acids; D010761:Phosphorus Radioisotopes; D014404:Tubulin; D006153:Guanosine Diphosphate; D008982:Molybdenum; D006160:Guanosine Triphosphate; D005990:Glycerol; C003125:phosphomolybdic acid