Tubulin polyglutamylase: partial purification and enzymatic properties.
In this work, we report on a novel enzyme, tubulin polyglutamylase, which catalyzes the posttranslational formation of polyglutamyl side chains onto alpha- and beta-tubulin. The length of the polyglutamyl side chain regulates the interaction between tubulin and various microtubule-associated proteins. We first developed an in vitro glutamylation assay. Activity measured in brain, a tissue particularly enriched with glutamylated tubulin, decreases during postnatal development. Thus, brains from 3-day-old mice were chosen as the starting material, and the enzyme was purified approximately 1000-fold. Its Mr was estimated to be 360K and its sedimentation coefficient 10 s. The enzyme catalyzes the MgATP-dependent addition of l-glutamate onto tubulin subunits. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl 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 some situations in vivo.
Regnard
C
C
Laboratoire de Biochimie Cellulaire, CNRS UPR 9065, Paris, France. regnard@ext.jussieu.fr
Audebert
S
S
Desbruyères
Denoulet
P
P
Eddé
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000201:enzymology; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D010453:Peptide Synthases; D011099:Polyglutamic Acid / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D050257:Tubulin Modulators
D014404:Tubulin; D050257:Tubulin Modulators; D011099:Polyglutamic Acid; D018698:Glutamic Acid; D012965:Sodium Chloride; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C112698:tubulin polyglutamylase