|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥1,800,000 (Direct Cost : ¥1,800,000)
1. Pulse Radiolysis Analysis of Electron Transfer Reactions of Cytochrome b561 : Pulse radiolysis analysis of electron transfer reactions of cytochrome b561 was performed. MDA radical oxidized rapidly (in msec time domain) the reduced heme b of cytochrome b561. In following time phase (in sec time domain), AsA re-reduced the oxidized heme b center. The rate of the oxidation of heme b with MDA radical showed a maximum at pH 5.5, whereas the reduction of heme b with AsA occurs at neutral pH. Further, only half of heme b could be reduced in the presence of excess MDA radical concentration.
2.Inhibition of Electron Transfer with DEPC-Treatment and Analysis of the DEPC-Modification Sites with MALDI-TOF-MS Spectrometry : Treatment of oxidized cytochrome b561 with DEPC, a reagent specific for HIS residue, caused an inhibition of electron transfer from AsA. Pulse radiolysis study showed that oxidation phase of reduced of heme b with MDA radical was similar to that observed for the control sampl
e. However, the following reduction phase with AsA was completely absent for the DEPC-treated sample. The modification sites were analyzed with MALDE-TOF-MS spectrometry using tryptic and V8 protease digests. Three major modification sites, His88, His161, and Lys85, were identified. It is considered that modification of His88 and His161, which were though as the ligands of heme b at the cytosolic side based on the membrane spanning model, caused the inhibition of the electron acceptance from AsA.
3. cDNA-cloning of Planarian Cytochrome b561. Planarian cytochrome b561 : cDNA was cloned and its deduced amino acid sequence was analyzed. Two fully conserved sequences were also conserved and were thought to be functionally essential. One of the 6 histidyl residues was found to be replaced with Asn residue. This lead to the suggestion that His88 and His161 are indeed the ligands of heme b at the cytosolic side.
4. cDNA-cloning of Arabidopsis thaliana Cytochrome b561 : Arabidopsis thaliana, a plant, showed to express a cytochome b561-like protein based on the EST-tag. We obtained two cDNA clones from an A.thaliana cDNA library. Analysis of the deduced amino acid sequences showed that the conserved sequence located at the intravesicular side, which which may play a role for the interaction with MDA radical, was well-conserved in plant also. The other conserved sequence for cytochrome b561 in animals was not conserved. In addition, one of the 6 conserved histiine residues showed a substitution to Gln. This residue corresponds indeed to the same site with the one where the substitution to occur in planarian cytochrome b561. Plant cytochrome b561 may evolved differently with a distinct physiological function from those of cytochrome b561 in animals. Less