Identification and kinetic characterization of HtDTC, the mitochondrial dicarboxylate-tricarboxylate carrier of Jerusalem artichoke tubers.
Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate-tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [14C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant Km and the Vmax at 25 degrees C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 microM and 10.9 micromol/(min mg protein), respectively; (b) the activation energy Ea of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between -5 and 35 degrees C. Similarly, the activation energy Ea of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between -2 and 35 degrees C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.
Spagnoletta
Anna
A
Dipartimento Farmaco-Biologico, Università della Calabria, Cosenza 87100, Italy.
De Santis
Aurelio
A
Tampieri
Elisabetta
E
Baraldi
Elena
E
Bachi
Angela
A
Genchi
Giuseppe
G
D000595:Amino Acid Sequence; D001692:Biological Transport; D003080:Cold Temperature; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry / Q000302:isolation & purification; D006368:Helianthus / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019802:Succinic Acid / Q000737:chemistry; D014233:Tricarboxylic Acids / Q000378:metabolism
D029382:Dicarboxylic Acid Transporters; D007656:Ketoglutaric Acids; D008081:Liposomes; D024101:Mitochondrial Proteins; D027361:Organic Anion Transporters; D014233:Tricarboxylic Acids; D019802:Succinic Acid