Tubulin polymerization with ATP is mediated through the exchangeable GTP site

Abstract

Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside kinase activity and 95% saturated with exchangeable GDP and nonexchageable GTP. All purine ribonucleoside 5′-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 μM and 7 μM, respectively. Most ribose- and polyphosphate-modified ATP analogs also su[[roted polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8- 14C]GDP bound in the exchangeable site of tubulin were displaced druing polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolognation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP: GTP ⪢ 1, but that GDP : ATP ⪡ 1. Inhibition appeared to be primarily competitive, since the higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site.