Via alternativa de transporte de elétrons em mitocôndrias de Vigna unguiculata (L.) Walp cv Vita 5: modulação pelo pH, pelo piruvato e pelo succinato

Abstract

All higher plants eontain a CN-resistant pathway catalyzed by the enzyme altemative oxidase whose function still remains unelear. Different mechanisms, based either on the influenee of the redox state of the enzyme or on its activation by aketo aeids control the altemative oxidase activity. In the present work, mitochondria were isolated (purified on Pereoll gradient) from etiolatecl hypocotyls of Vigna unguiculata cv. Vita 5 (susceptible to salt stress) and the electron altemative pathway was measured in the presenee of cyanide and antimyein A. <h uptake was polarographycally estimated. The presenee of the altemative oxidase was detected by monoclonal antibodies against altemative oxidase proteins of Sauromatum guttatum and revealed two forms of the enzyme under 35 and 70 kDa. The effect of pH on the CN-resistant respiration was investigated in a pH range from 5.75 to 8.25 with NADH as substrate, in the presenee of DlT. The highest rate of CN-resistant respiration was obtainecl at pH 6.25. Inerease in pH over 6.25 and decrease below 6.25 resultecl in progressive decrease in O2 uptake. It has also been demonstratecl the stimulatory effect of inereasing concentrations of pyruvate (until 500 J.1M) on the CN-resistant respiration at pH 6.25, 7.25 and 7.75. The highest stimulation of CNresistant respiration by pyruvaate was observed at pH 7.25. The pH effect on CN-resistant respiration was shown to be the same in the presenee of nigeriein, an ionophore known to abolish (pH between matrix and intermembrane sides. The reversion of reaction medium pH (6,25 to 7,75 and 7,75 to 6,25) also indueed a reversibility in the effect of pH and pyruvate on the CN-resistant respiration. The pH effect on the altemative oxidase activity was tentatively attributed to a new status of this protein namely a protonated/deprotonated status according to a scheme in which the most active form is H+Ered-Pir followed by H+Ered.