Isolation of tubulin polyglutamylase from Crithidia; binding to microtubules and tubulin, and glutamylation of mammalian brain alpha- and beta-tubulins.
Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzymatic activity that incorporates radioactive glutamic acid in a Mg2+-ATP-dependent manner into alpha- and beta-tubulins. The tubulin polyglutamylase is extracted by 0.25 M salt. The Crithidia enzyme can be purified by ATP-affinity chromatography, glycerol-gradient centrifugation and ion-exchange chromatography. After extraction from the microtubular cytoskeleton the glutamylase forms a complex with alphabeta tubulin, but behaves after removal of tubulin as a globular protein with a molecular mass of 38x10(3). In highly enriched fractions a corresponding band is the major polypeptide visible in SDS-PAGE. The enzyme from Crithidia recognises mammalian brain tubulin, where it incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins. Synthetic peptides with an oligoglutamyl side chain, corresponding to the carboxy-terminal end of brain alpha- and beta-tubulins, are accepted by the enzyme, albeit at low efficiency. The polyglutamylase elongates the side chain by up to 3 and 5 residues, respectively. Other properties of the tubulin polyglutamylase are also discussed.
Westermann
S
S
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11, Germany.
Schneider
A
A
Horn
E K
EK
Weber
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D003421:Crithidia / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007891:Leishmania / Q000201:enzymology; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases; D011099:Polyglutamic Acid / Q000302:isolation & purification* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D010446:Peptide Fragments; D014404:Tubulin; D011099:Polyglutamic Acid; D010453:Peptide Synthases; C112698:tubulin polyglutamylase