Spatial separation of the two essential thiol groups and the binding site of the exchangeable GTP in brain tubulin

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu²⁺ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by $\text{N-}\text{ethylmaleimide}$, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg²⁺, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of $\text{N-}\text{ethylmaleimide}$. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn²⁺ for Mg²⁺. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups, b. The binding sites of the exchangeable GTP and Mg²⁺ are located 10 Å, at least, from the two most reactive sulfhydryl groups, c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 Å. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.