Identification and kinetic characterization of HtDTC, the mitochondrial dicarboxylate-tricarboxylate carrier of Jerusalem artichoke tubers

摘要: Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant cold and freezing. The aim of this study was perform a kinetic characterization the mitochondrial dicarboxylate–tricarboxylate carrier (HtDTC) assess possible involvement in tolerance tubers. HtDTC purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite Matrex Gel Orange A. SDS gel electrophoresis fraction showed single polypeptide band with an apparent molecular mass 31.6 kDa. A polyclonal antibody raised against tobacco DTC cross-reacted protein Western blot analysis. In trypsin, digestion yielded peptides that exhibited strong amino acid sequence similarity previously identified plant DTCs. Furthermore, using degenerate primers, portion Htdtc cDNA amplified sequenced; encoded for high known homolog When reconstituted liposomes loaded dicarboxylate (2-oxoglutarate, malate, malonate, succinate, maleate) or tricarboxylate anions (citrate, trans-aconitate, isocitrate), transported all these exchange external [14C]2-oxoglutarate. performed: (a) half-saturation constant Km Vmax at 25∘C 2-oxoglutarate/2-oxoglutarate found 360 μM 10.9 μmol/(min mg protein), respectively; (b) activation energy Ea succinate/2-oxoglutarate 50.7 kJ/mol between −5 35∘C. Similarly, succinate respiration mitochondria, measured −2 35∘C, shown (65.3 kJ/mol). physiological relevance properties temperature dependence transport activities is discussed respect ability