Release of exchangeably bound guanine nucleotides from tubulin in a magnesium-free buffer

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Abstract
The number of moles of guanine nucleotides (denoted GXP), either GTP or GDP, bound to a mole of phosphocellulose-purified tubulin after gel filtration into a variety of nucleotide-free buffers was measured. All buffers that promote reduction of the number of bound nucleotides to fewer than 2/tubulin dimer also eventually cause irreverisble loss of activity of the protein. In 0.1 M 1,4-piperazinediethanesulfonic acid (pH 6.9) and 2 mM dithioerythritol (with no Mg2+), tubulin rapidly releases approximately 0.4 mol of bound nucleotides during 2 successive gel filtrations requiring less than 0.5 h and regains the ability to polymerize when Mg and GTP are immediately added to the buffer. No change in conformation detectable by circular dichroism or sedimentation velocity accompanies this reversible process. (Upon prolonged incubation in the buffer tubulin undergoes irreversible changes according to apparent 1st-order kinetics with a half-life of .apprx. 8 h. These changes include the irreversible release of nucleotide, a loss of the ability to polymerize, and a decrease in molar ellipticity between 210 and 240 nm.) The nucleotide which is reverisbly released in this buffer comes from that population which exchanges readily with [3H]GTP in vitro. When tubulin purified by phosphocellulose chromatography is incubated in a Mg2+-containing buffer for 30 min with [3H]GTP at concentrations from 70 .mu.M to 1 mM at temperatures from 0-24.degree. C, the difference between the total GXP bound and the [3H]GTP that becomes bound by exchange is consistently 1.2 .+-. 0.1 per tubulin dimer. Phosphocellulose-purified tubulin is evidently composed of 2 types of dimers: one (.apprx. 80% of the total) which readily exchanges guanine nucleotides at its exchangeable site and one (.apprx. 20% of the total) which exchanges nucleotides poorly. Those dimers that exchange GTP readily in Mg2+-containing buffers appear to be the same dimers that rapidly release bound nucleotide in Mg2+-free buffers.

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