Carboxy-ethyl-N-phenylcarbamate was synthetized as described in Mizuno et al. (1981), and coupled to sepharose 4B (Amersham-Pharmacia, Freiburg, Germany) that had been extended by an aminoethyl linker (Cuatrecasas, 1970). Coleoptile segments were ground by mortar and pestle in liquid nitrogen and a soluble extract produced and fractionated as described in Freudenreich and Nick (1998) using a microtubule-stabilizing buffer (MSB: 25 mm Mes, 5 mm EGTA, 5 mm MgCl2, 1 m glycerol, 1 mm GTP, 1 mm DTT, 1 mm phenylmethylsolphonyl fluoride, 1 µg ml−1 leupeptin, pepstatin and aprotinin, pH 6.9). For small samples, the sepharose was filled onto glass wool into 50-µl Eppendorf tips and fractions were collected by centrifugation (1 min, 15 000 g) into small tubes. The fractions were precipitated by trichloroacetic acid (Bensadoun and Weinstein, 1976) prior to processing for SDS-PAGE. For the assembly of microtubules from tyrosinylated tubulin, the fractions were concentrated by ultrafiltration (Centrex UF-2, Schleicher and Schüll, Dassel, Germany) to 1 mg ml−1 of total protein. Microtubule assembly was induced in these samples as described in Nick et al. (1995). The microtubules were treated with 0.1 units ml−1 carboxypeptidase A (C9268, Sigma-Aldrich, Neu-Ulm, Germany) in MSB at 30°C for variable time intervals (0–60 min), the reaction was stopped by transfer to ice and the carboxypeptidase A was washed out by collecting the microtubules by ultracentrifugation (300 000 g, 10 min, 4°C), replacing the supernatant by the same volume of fresh MSB and repeating this step. Then, the microtubule sediment was solubilized on ice by 1 mm CaCl2 in MSB by using a glass rod for 15 min, the sediment was removed by ultracentrifugation (300 000 g, 10 min, 4°C) and the supernatant was assayed by EPC sepharose chromatography. The C-terminal peptide of maize TUBA2 was synthetized (Pepscan Systems, Lelystad, the Netherlands) either in the tyrosinylated form (FDEGEEGDDGDEY) or in the de-tyrosinylated form (FDEGEEGDDGDE) and conjugated to ovalbumin as a carrier via N-terminal cysteine. The peptide was used at 0.5 µg µl−1 (in MSB) for EPC sepharose chromatography. In some experiments, the tyrosinylated conjugate was pre-treated with 0.1 units ml−1 carboxypeptidase A (60 min, 30°C) and purified on a sephadex G-25 column prior to EPC sepharose chromatography.