Incorporation of nitrotyrosine into alpha-tubulin by recombinant mammalian tubulin-tyrosine ligase.
Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.
Kalisz
H M
HM
Gesellschaft für Biotechnologische Forschung, Abteilung Zellbiologie, Braunschweig, Germany.
Erck
C
C
Plessmann
U
U
Wehland
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010453:Peptide Synthases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014404:Tubulin / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D011994:Recombinant Proteins; D014404:Tubulin; C002744:3-nitrotyrosine; D014443:Tyrosine; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase