Structure and function of membrane-spanning cytochrome b561 in neuroendorine secretory vesicles
Grant-in-Aid for Scientific Research (C)
|Research Institution||Himeji Institute of Technology|
TSUBAKI Motonari Himeji Institute of Technology Associate Professor Faculty of Science, 理学部, 助教授 (00145046)
|Project Fiscal Year
1996 – 1997
Completed(Fiscal Year 1997)
|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1997 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1996 : ¥2,300,000 (Direct Cost : ¥2,300,000)
|Keywords||vitamin C / ascorbic acid / cytochrome b561 / noradrenaline / catecholamine / chromaffin vesicle / adrenal medulla / heme / ビタミンC / アスコルビン酸 / シトクロムb_<561> / ノルアドレナリン / カテコール アミン / クロマフィン小胞 / 副腎髄質 / ヘム / カテコールアミン|
1.Purification Procedure of Cytochrome b561
After the purification of chromaffin vesicles from bovine adrenal medulla, the vesicle membranes were solubilized with 1% b-octy1 glucoside in the presence of ascorbic acid (AsH). Cytochrome b561 was purified to homogeneity with w-aminoocty1 Sepharose 4B column chromatographies.
2.Heme Content Analysis of Purified Cytochrome b561
The heme content analysis of the purified cytochrome b561 with a pyridine hemochrome method showed a presence of about 1.7 heme B molecules per cytochrome b561 monomer.
3.EPR Analysis of the Purified Cytochrome b561
EPR spectroscopy revealed a presence of three distinct low-spin heme species which showed redox-dependent spectral changes upon ferricyanide oxidation. These low-spin heme species are likely derived from the two independent heme B centers in cytochrome b561.
4.Selective Inactivation of Cytochrome b561 with a Mild Alkaline-or a DEP-Treatments
Treatments of the purfied cytochrome b561 in oxidized atate with a mild alkaline condition or a low DEP concentration caused a selective loss of the ability to reseive electron equivalent from ascorbic acid for about a half of the heme centers.
5.Analysis of Electron Transfer Mechanism of Cytochrome b561 with pulse Radiolysis Method
We analyzed electron transfer reaction between reduced cytochrome b561 and monodehydroascorbic acradical (MDA) which produced by a pulse radiolysis method.We found that the oxidation with MDA and the reduction with AsH occur in different heme centers of cytochrome b561.
6.A Membrane-spanning Model of Cytochrome b561
Based on these results, we proposed a membrane-spanning model of cytochrome b561. The two heme centers are located at cytosolic and intravesicular sides, respectively, in this model. Since the two completely conserved sequences are located near these heme binding regions, these two conserved sequences may from an AsH-binding site at cytosolic aide and an MDA-binding site at intravesicular side, respectively.
Research Output (14results)