Protein-protein interaction of cytochrome b561 in chromaffin vesicle membranes studied by two-dimensional blue-native/sodium dodecyl sulfate gel electrophoresis and co-immunoprecipitation analysis

Takeuchi, Fusako: Protein-protein interaction of cytochrome b561 in chromaffin vesicle membranes studied by two-dimensional blue-native/sodium dodecyl sulfate gel electrophoresis and co-immunoprecipitation analysis. Acta biologica Szegediensis, (50) 1-2. pp. 83-87. (2006)

[img] Cikk, tanulmány, mű
5083.pdf

Download (533kB)

Abstract

We analyzed a protein-protein interaction in solubilized chromaffin vesicles using two-dimensional electrophoresis (1st, Blue-Native PAGE; 2nd, Tricine-SDS-PAGE). Cytochrome b561 band, which was verified by immunoblotting, was observed in the two-dimensional gel with an apparent molecular weight of ~100~400kDa. On the other hand, purified cytochrome b561 showed a monomeric band (28 kDa) in Blue-Native PAGE. These results indicated that cytochrome b561 interacts with other proteins in the chromaffin vesicles to form a large protein complex(es). To clarify the nature of the interaction, we performed co-immunoprecipitation experiments, where the solubilized membrane proteins were treated with immunopurified anti- b561 IgG antibodies followed by sedimentation with protein-A-Sepharose. We found that there were no other proteins co-sedimented with cytochrome b561. Since the immunopurified anti- b561 IgG antibodies bound specifically to the C-terminal hydrophilic portion of cytochrome b561 protein, we concluded that such binding of the IgG antibodies to the C-terminal portion might cause an inhibition of protein-protein interaction with other proteins in the solubilized state.

Item Type: Article
Event Title: International Conference on Membrane Redox Systems and Their Role in Biological Stress and Disease, 8., 2006, Szeged
Journal or Publication Title: Acta biologica Szegediensis
Date: 2006
Volume: 50
Number: 1-2
Page Range: pp. 83-87
ISSN: 1588-385X
Language: angol
Heading title: Proceedings
Uncontrolled Keywords: Természettudomány, Biológia
Additional Information: Bibliogr.: 87. p.; Abstract
Date Deposited: 2016. Oct. 17. 09:24
Last Modified: 2018. May. 25. 13:14
URI: http://acta.bibl.u-szeged.hu/id/eprint/22776

Actions (login required)

View Item View Item