TUBULIN POLYGLUTAMYLASE - PARTIAL-PURIFICATION AND ENZYMATIC-PROPERTIES

In this work, we report on a novel enzyme, tubulin polyglutamylase, wh ich catalyzes the posttranslational formation of polyglutamyl side cha ins onto alpha-and beta-tubulin. The length of the polyglutamyl side c hain regulates the interaction between tubulin and various microtubule -associated proteins. We first developed an in vitro glutamylation ass ay. Activity measured in brain, a tissue particularly enriched with gl utamylated tubulin, decreases during postnatal development. Thus, brai ns from 3-day-old mice were chosen as the starting material, and the e nzyme was purified similar to 1000-fold. Its M-r was estimated to be 3 60K and its sedimentation coefficient 10 s. The enzyme catalyzes the M gATP-dependent addition of L-glutamate onto tubulin subunits. Microtub ules are much better substrates than unpolymerized tubulin, and the re action is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added seq uentially onto tubulin, leading to progressive elongation of the polyg lutamyl side chains. Side chains of one to six or seven glutamyl units were obtained with microtubules, whereas much longer side chains (up to 15-20 units) were formed with unpolymerized tubulin. Interestingly, such very long polyglutamyl side chains were recently detected in som e situations in vivo.