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 am ino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with al pha -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 fr om different organs of mung bean seedlings, we find that there are two hybr idizable AOX bands in mitochondria. Their molecular weights are about 35 an d 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, t rue leaf and cotyledon of mung bean seedlings show that true leaf has the h ighest total respiration (V-1), alternative pathway (AP) capacity (V-alt) a nd the activity of AP (pV(alt)) among the three organs. V-t and pV(alt) of cotyledon ranked the second. Hypocotyl has the lowest V-t and pV(alt), but its V-alt is higher than that of cotyledon. These results are consistent wi th the analysis of Western blotting of expression of AOX. The highest V-t a nd pV(alt) in true leaf are accompanied two hybridizable polypeptides of AO X protein, 35 ku and 38 ku respectively. The next is cotyledon V-t and pV(a lt) with only one 38 ku hybridizable polypeptide of AOX protein. Hypocotyl V-t and pV(alt) is the lowest and its immunoblotting band is similar to tha t of cotyledon, but the expressive amount of 38 ku protein is less than tha t of cotyledon. The results suggest that the 35 ku AOX may contribute mainl y to true leaf pV(alt).