Relationship between tubulin SH groups and bound guanine nucleotides

Guanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55,000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8% and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Furthermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.