A brain protein (P30) that immunoreacts with a polyclonal anti-pancreatic carboxypeptidase A antibody shows properties that are shared with tubulin carboxypeptidase.

A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 +/- 0.2). When the tubulin carboxypeptidase preparation was subjected to gel filtration chromatography under low salt concentration, P30 behaved as a protein of molecular mass 38 kDa whereas tubulin carboxypeptidase eluted at a position of 75 kDa molecular mass. However, when the chromatography was performed at relatively high salt concentration they behaved as proteins of 49 and 56 kDa, respectively. We considered that P30 may be an inactive monomeric form of the dimeric tubulin carboxypeptidase. However we can not rule out the possibility that it represents another carboxypeptidase not yet described.

Immunodetection of tubulin carboxypeptidase activity on nitrocellulose membrane after gel electrophoresis and blotting.

It was found that the detyrosination of tyrosinated tubulin by tubulin carboxypeptidase can occur when both the enzyme and the substrate are adsorbed on nitrocellulose. This, and the use of a specific antibody that recognizes detyrosinated tubulin allowed us to localize tubulin carboxypeptidase on a nitrocellulose membrane after agarose gel electrophoresis and blotting. The method was also extended to detect pancreatic carboxypeptidase A.

The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase.

Tubulin dimer, alpha-tubulin subunit, and C-terminal peptides obtained from the alpha-tubulin subunit were compared in their capabilities to act as substrates of tubulin carboxypeptidase. The results obtained indicate that the enzyme does not require the beta-tubulin subunit to release tyrosine from alpha-tubulin. The 17-Kd C-terminal peptide of the alpha-tubulin subunit was obtained and it was detyrosinated at the same rate as tubulin dimer. A smaller C-terminal peptide of 2.8-3.7 Kd showed a lower capability to act as substrate. Similar results were obtained with pancreatic carboxypeptidase A. From the analysis of the results we consider that an optimal activity of the tubulin carboxypeptidase depends mainly on the accessibility of the C-terminal end of alpha-tubulin.