PURIFICATION OF A MUNG BEAN PROTEIN-BINDING TO MICROTUBULES THROUGH 2DEFINED SITES IN THE CARBOXYL-TERMINAL DOMAIN OF BETA-TUBULIN

A protein purified from mung beans binds to monomeric tubulin and micr otubules through two sites near the C-terminus of beta-tubulin. The 34 -kDa protein cosediments with taxol-stabilized brain microtubules and is purified by ion-exchange chromatography and two steps of affinity c hromatography using tubulin and S-tubulin (tubulin cleaved with subtil isin at Gln-433). The 34-kDa protein binds to tubulin but not to S-tub ulin whether monomeric or polymeric. A rabbit antiserum was raised aga inst a synthetic peptide of 11 amino acids corresponding to beta(422-4 32), the highly conserved region which lies just outside the subtilisi n-cleaved C-terminal peptide. Partially-purified antibodies were used to generate murine anti-idiotypic monoclonal antibodies. One monoclona l antibody, which does not bind to sheep brain or mung bean tubulin, r eacts in ELISA's with microtubule-associated proteins from the brain a nd with the 34-kDa protein from mung beans. On immunoblots, the anti-i diotypic antibody recognizes several bands two of which have similar M (r) values to previously reported plant microtubule-associated protein s. The results indicate that the mung bean protein, like neuronal MAP- 2 and tau-protein, binds to both the variable, (highly acidic region c leaved by subtilisin) and to the highly conserved beta(422-432) sequen ce. We conclude that the 34-kDa protein is likely to be a plant MAP th at binds to a site known to be important in the regulation of tubulin polymerization. Anti-idiotypic antibodies and tubulin/S-tubulin affini ty chromatography may have wide applicability in the identification of plant MAPs.