| Abstract: | Tubulin was isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose) and ion exchange (DEAE-Sephacel) chromatography from mung bean and cultured carrot suspension cells. SDS-PAGE (Blose 1981) of mung bean tubulin has shown it to consist of two major subunits (MBT1 and MBT2) and a minor subunit (MBT3). Tubulin isolated from carrot cells was resolved into only two bands on SDS-PAGE (slow moving subunit was named CT1). However, the faster moving subunit on SDS-PAGE was resolved into two bands (CT2 and CT3) on SDS-4M urea-PAGE. On SDS-4M urea-PAGE, CT1 migrated faster than CT2, CT3. By contrast in SDS-4M urea-PAGE, mung bean tubulin remains unresolved. Mammalian tubulin could be resolved into alpha and beta-subunits in both electrophoretic systems. Monoclonal antibodies to mammalian alpha and beta-tubulin subunits (MCA-T alpha and MCA-T beta, respectively) and Western blot analysis clearly demonstrated a cross-reactivity of MCA-T alpha with MBT2, MBT3, CT2 and CT3, while MCA-T beta showed cross-reactivity with MBT1 and CT1. Although MBT2, MBT3, CT2 and CT3 are immunologically related to the alpha-subunit of mammalian tubulin, their migration on SDS-PAGE was reversed with respect to MBT1 or CT1, which were immunologically related to the beta-subunit of mammalian tubulin. Peptide mapping patterns also supported above the results. |
