Studies on the relationship between cyanide-resistant respiration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with alpha-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (V(t)), alternative pathway (AP) capacity (V(alt)) and the activity of AP (rhoV(alt)) among the three organs. V(t) andrhoV (alt) of cotyledon ranked the second. Hypocotyl has the lowest V(t) andrhoV (alt), but its V(alt) is higher than that of cotyledon. These results are consistent with the analysis of Western blotting of expression of AOX. The highest V(t) andrhoV (alt) in true leaf are accompanied two hybridizable polypeptides of AOX protein, 35 ku and 38 ku respectively. The next is cotyledon V(t) andrhoV (alt) with only one 38 ku hybridizable polypeptide of AOX protein. HypocotylV (t) andrhoV (alt) is the lowest and its immunoblotting band is similar to that of cotyledon, but the expressive amount of 38 ku protein is less than that of cotyledon. The results suggest that the 35 ku AOX may contribute mainly to true leafrhoV (alt).