Structure of tubulin C‐terminal domain obtained by subtilisin treatment The major α and β tubulin isotypes from pig brain are glutamylated

Abstract

Limited ~ubtihsin digestion of the tubulin ~, fl heterodlmei ha~ been used in this ~vork to reduce the total number of tubulin isotypes from 20 for native to 9 re1 bubtdma.¢lcaved tubuhn This indicalc~ that ~he major part of tul~uhn h,*terogenelty is located at the C.terminus of the molecule. The C-terminal pcpfides of both a and fl ~ubumt~ of tt,buhn ~.ere purified by anion-enchange HPLC. Combined tt~e of Edman desradation chemistry and mass spectrometry on the isolated peptlde~ shown that subtdi~in cleavase occurs at posmon Asp-438 and His-406 of ct and Gin-433 and His.396 o1"/3 tubuhn chums. Quaiahtat~ve analysis of our data show that cleavage at po~it~ons His.406 (~) and I-1is-396 (.8) occurs with a low efficient), an~l indicates that the naajor i~otylgeS of P~8 brain tubulm are modified by sequemial altachmeat of I to 5 glutam~c acid residues at posit~ons or -435 of ¢ and/3 tubuhn, respectively

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

“…Shown in Figure 2a is a representative ESI mass spectrum acquired for an aqueous ammonium acetate solution (100 mM, pH 6.8) of αβ-tubulin (14 μM). Abundant ion signal corresponding to tubulin dimer (d n+ ), at charge states +18 to +22, is evident; low abundance signal corresponding to tubulin tetramer ions, at charge states +32 to +29, was also [48,49] are in agreement with theoretical values (UniProt, P02550 for αI and P02554 for βI) of 101,677 Da and 203,354 Da, respectively. The presence of tetramer could be due to selfassociation of the tubulin dimer; nonspecific binding during the ESI process might also play a role in its formation [38,39].…”

Probing the Kinesin-Microtubule Interaction

“…The work reported here is the first attempt to map directly the sites of interaction between microtubules and the kinesin motor; these results point to the C termini of ␣-and/or ␤-tubulin as sites where binding may occur. Furthermore, treatment of paclitaxel-stabilized microtubules with the protease subtilisin, which is reported to remove C-terminal fragments from the ␣-and ␤-tubulin subunits (30), increased the K m for ATPase activation of the kinesin motor while the V max remained unchanged. One interpretation is that subtilisin removes one of the kinesin binding sites on ␣-or ␤-tubulin, but that additional sites exist.…”

Reversible polyglutamylation of alpha- and beta-tubulin and microtubule dynamics in mouse brain neurons.

“…This finding has been extended to class III (a neuron-specific f3-tubulin isotype, denoted here d3') (Alexander et al, 1991) and class II (Redeker et al, 1992;Riidiger et al, 1992) 0-tubulin. Polyglutamylated tubulin is very abundant in nerve cells and is also found, at basal levels, in other cell types (Edde et al, 1990;Wolff et al, 1992).…”

“…Indeed, glutamylation is a polymodification, and several tubulin species bearing one to at least six negatively-charged glutamyl units were characterized (Edde et al, 1990;Alexander et al, 1991;Redeker et al, 1992;Rudiger et al, 1992). Because the GT335 mAb recognizes indifferently all of these species (Wolff et al, 1992), it discriminates between glutamylated and nonglutamylated forms but cannot reveal a shortening or extension of the polyglutamyl chain.…”

“…[3H]glutamate, as well as immunoblotting experiments with GT335, led to the detection of acidic variants of class III (p) and nonclass III fl-tubulin, the glutamylation of which has been recently demonstrated (Alexander et al, 1991;Redeker et al, 1992;Rudiger et al, 1992;Wolff et al, 1992).…”