The effects of elevated pH and high salt concentrations on tubulin

Abstract

The effects of incubating phosphocellulose-purified bovine tubulin at 4 °C in nucleotidefree buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH 4) 2SO 4, or NH 4Cl have been studied. At pH ⩾ 7.5 or at NaCl concentrations ⩾ 0.7 m, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 m 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 m NH 4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 m (NH 4) 2SO 4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220–260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 m m guanosine 5′-triphosphate (GTP) or 4 m glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 m NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 m NaCl is decreased in the presence of 1 m m GTP, and tubulin is protected almost completely from 1 m NaCl-induced loss of GTP (and retains the ability to exchange [ 3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.