2003 Fiscal Year Final Research Report Summary
Reconstitution of neuroendocrine vesicles by utilizing proteoliposomes and chromaffin vesicles
| Project/Area Number |
14580676
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Kobe University |
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Principal Investigator |
TSUBAKI Motonari Kobe University, Graduate School of Science and Technology, Professor, 大学院・自然科学研究科, 教授 (00145046)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Ascorbic acid / Cytochrome b561 / Electron transfer / Heme protein / Membrane protein / Dopamine β-hydroxylase / Neuroendocrine vesicle / Catecholamine |
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| Research Abstract |
Cytochrome b_<561> from chromaffin vesicles contains two hemes b with different midpoint potentials and participates in transmembrane electron transport from extravesicular ascorbate (AsA) to an intravesicular monooxygenase, dopamine β-hydroxylase. Treatment of oxidized b_<561> with diethylpyrocarbonate (DEPC) caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of AsA during the treatment. EPR analyses showed that, upon the treatment, the g_z=3.69 heme species was converted to a non-AsA-reducible form, although its g_z value showed no appreciable change. The treatment had no effect on the other heme (the g_z=3.13 species). The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the DEPC-treated b_<561> when ASA was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of AsA and increases both the reduction
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rate and the final reduction level of the heme center on the intravesicular side of the DEPC-treated cytochrome. The purified b_<561> was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When AsA-loaded vesicles with b_<561> were mixed with ferricytochrome c, the intravesicular AsA was able to reduce external thiazole blue or cytochrome c. The topology of the reconstituted b_<561> in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that b_<561> was reconstituted into the membranes in an inside-out orientation irrespective of the modification with DEPC. The addition of a soluble form of dopamine β-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon addition of ferricyanide as a mediator between b_<561> and dopamine β-hydroxylase. Less
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