Monoclonal Antibodies as Tools for the Identification of the Tonoplast Sucrose Carrier from Sugarcane Stalk Tissue

Abstract

Poly- and monoclonal (IgM) antibodies were prepared against tonoplast preparations from immature sugarcane stalks and the antibodies were tested for the ability to inhibit sucrose transport. Sucrose transport with and without MgATP present was strongly inhibited. Nitrate-sensitive ATPase activity and nonspecific PPiase activities were not affected. The inhibitory effect of the antibodies was measured in the absence of permeable ions and under conditions where the anion channel blocker DIDS has no effect on sucrose transport. Inhibition of sucrose transport by mAB C50-5-3 could be counteracted by high sucrose concentrations. The sugarcane antibodies cross-reacted with tonopasss prepared from mature red beet storage roots. The proteins from tonoplast vesicles of sugarcane stalk tissue were solubilized with octyl glucoside or Triton X-100 and were separated by size exclusion chromatography. ELISA-assays with FPLC purified IgM antibodies of fractions from FPLC gel filtration of solubilized tonoplast proteins revealed an active 55 kDa fraction. Proteoliposomes prepared from this fraction took up sucrose at a 48fold higher rate than native sugarcane tonoplast vesicles. These mAbs appear suitable for future use in purifying the tonoplast sucrose carrier.