Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects.

J L Fishback,L R Yarbrough

doi:10.1016/s0021-9258(17)43502-2

Abstract

In the presence of glycerol, the thionucleotide 2-amino-6-mercapto-9-ribofuranosyl purine 5'-triphosphate (S6-GTP) promotes the assembly of 6 S tubulin to form microtubules. Microtubules assembled with this analog show normal stability properties. In the absence of glycerol, few microtubules are formed with S6-GTP; however, many twisted ribbons are evident. Binding of S6-GTP to tubulin from which the associated proteins and exchangeable nucleotide have been removed (Tu(-] produces about a 16% quenching of intrinsic tubulin fluorescence. Fluorescence titrations indicate an apparent Kd for the tubulin S6-GTP complex of about 3 X 10(-8)M. Binding of S6-GTP to Tu(-) also produces a change in its absorption spectrum. The observed difference spectrum has a maximum at 350 nm and negative extrema at 323 and 338 nm. This suggests that the environment of the thioguanine ring is relatively hydrophobic. Competitive displacement studies yield apparent Kd values of about 1.7 X 10(-8)M for GTP and 8.3 X 10(-8) M for GDP. The changes in absorbance and fluorescence which accompany binding provide an excellent approach to the study of the kinetics and mechanisms of nucleotide binding as well as studies of the kinetics of displacement of GTP, GDP, and their analogs.

Highlights

In the presence of glycerol,the thionucleotide 2- the bound GTP is not hydrolyzed during reassembly of miamino-6-mercapto-9-ribofuranosyplurine 5’-triphos- crotubules [3,4,5]
Fluorescence titrations tightly bound GDP is firrsetmoved [7,8,9,10,11]. Such observations indicate an apparent Kd for the tubulin S6-GTP com- led Kirschnerandhis colleagues to suggest that guanine plex of about 3 x lo-’ M
Binding of S6-GTP to Tu(-) nucleotides acted as allostericeffectors to promote tubulin produces a change in its absorption spectrum

Summary

EQUILIBRIUM ASPECTS*

Observed difference spectrum has a maximum at 3 5 0 As part of our studies on the molecular mechanisms innm and negativeextremaat 323 and 338 nm This volved in the promotion of tubulin reassembly by GTP, we suggests that the environment of the thioguanine ring have attempted todevise approaches to examine theequilibis relatively hydrophobic. Theseanalogs, which contain a absorbanceand fluorescence which accompanybinding blocked phosphoryl terminus, bindrelatively tightly and speprovide an excellent approach to the study of the ki- cifically to the tubulin E site; they do not support netics and mechanisms of nucleotide binding as well as tubulin reassembly. Guanine nucleotide-binding site on tubulin at which exchange occurs; PIPES, piperazine-N,N’-bis(2-ethanesulfonicacid); MES2, -(Nmorpho1ino)ethanesulfonic acid; MAPS, microtubule-associated proteins; Tu, tubulin; EF, elongation factor; Tu(-), tubulin from which

MATERIAL SA N DM ETHODS

Findings

Microtubules were reassembled in