|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥1,300,000 (Direct Cost : ¥1,300,000)
A trapping reagent for superoxide anion(O_<2^->) was newly developed by stabilizing an adduct of 0_<2^-> with peroxidase, in which the stablization of the adduct was achieved by introducing the electron withdrawing side chains to the heme in peroxidase. The ability of the O_<2^-> trapping reagent was assessed by analyzing the oxygen metabolite in the respiratory burst of neutrophils. The results indicated :
1)In the respiratory burst voked by soluble stimuli, the most of consumed oxygen was converted to 0_<2^->.
2)During the phagocytosis, the O_<2^-> formation was found only in the intial stage of the reaction, and with the phagosome formation, hydrogen peroxide was detected in the outside of the cell. These indicated that the newly developed method was the most powerful tool for O_<2^-> detection. We also found that cytochrome b_<558> acted as the terminal oxidase in the respiratory burst of neutrophils.
By using the new method, the oxygen metabolism of thyroid cells was examined and summarized below.
1)The isolated plasma membrane exhibited NADPH-supported burst respiration with a concomitant release of O_<2^->.
2)On the other hand, whole cells released hydrogen peroxide, not O_<2^->, upon addition of CA^<2+>. these suggested that hydrogen peroxide was produced in the cytosol from O_<2^->by the action of superoxide dismutase, and was released out of cell. We also tried to analyze the oxygen metabolizm of sea urchin egg, but the activity of oxygen consumption was very low. Further study was required for the elucidation of the oxygen metabolism.
We also attempt to elucidate whether nitric oxide, a newly discovered signal transducing substance, plays a orle for the activation of burst respiration. For the purpose, the measurement of nitric oxide in n mole order by using soluble genylate cyclase is planned, and we succeeded to purify the enzyme form boving lung.