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Triton X-114を用いるミセル水性二相分配法による膜タンパク質の抽出に及ぼす荷電水溶性ポリマーの添加効果

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Title: Triton X-114を用いるミセル水性二相分配法による膜タンパク質の抽出に及ぼす荷電水溶性ポリマーの添加効果
Other Titles: Effect of charged water-soluble polymers on the extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system
Authors: 谷, 博文1 Browse this author →KAKEN DB
大浦, 貴史2 Browse this author
上舘, 民夫3 Browse this author
Authors(alt): Tani, Hirofumi1
Ooura, Takashi2
Kamidate, Tamio3
Keywords: charged polymer
Triton X-114
two-phase separation
membrane protein
Issue Date: Dec-1998
Publisher: 日本分析化学会
Journal Title: 分析化学
Volume: 47
Issue: 12
Start Page: 965
End Page: 970
Publisher DOI: 10.2116/bunsekikagaku.47.965
Abstract: The effect of charged water-soluble polymers on the extractability of membrane proteins in a Triton X-114-based aqueous micellar two-phase system was examined. Cytochrome b5 (Cyt.b5), NADH-cytochrome b5 reductase (fp1), and NADPH-cytochrome P450 reductase (fp2), used as model proteins, were to well-extracted into a surfactant-rich phase without polymers. In the presence of non-charged dextran, there were no changes in their extractability. However, by the addtion of cationic diethylaminoethyl-dextran at the concentration range of 0.01 ~ 0.3%, the extractability of Cyt.B5 and fp2 was greatly reduced. In contrast, the use of anionic dextran sulfate at the same concentration range depressed the extraction of fp1 alone. A further increase in the polymer concentration or salt addition resulted in an increase in the extractability. From a calculation of net charge of the proteins, it was shown that Cyt.b5 and fp2 are negative and fp1 is positive at pH 7.4, where the extraction was conducted. Thus, the depressed extraction of the proteins in the presence of the charged polymers is attributable to the electrostatic interaction between the proteins and the charged polymers in the aqueous phase. These results indicate that charged polymers could be effective for the selective extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system.
Triton X-114 を用いたミセル水性二相分配系に荷電水溶性ポリマーを添加し,膜タンパク質の界面活性剤相への抽出に及ぼす影響を検討した.膜タンパク質にはミクロゾーム中で電子伝達系を構成するチトクロムb5,NADH-チトクロムb5還元酵素,NADPH-チトクロムP450還元酵素を用いた.電荷を持たないデキストランを添加した場合,添加しなかったときと同様,いずれの膜タンパク質も界面活性剤相に抽出された.一方,正電荷を持つジエチルアミノエチルデキストランを添加すると,チトクロムb5及びNADPH-チトクロムP450還元酵素の抽出が,負電荷を持つ硫酸デキストランを用いるとNADH-チトクロムb5還元酵素の抽出がそれぞれ抑制された.膜タンパク質の電荷並びに塩を添加したときの抽出結果から,荷電ポリマーによる抽出抑制効果は膜タンパク質と水相中のポリマーとの静電的相互作用によることが明らかになった.
Type: article
Appears in Collections:工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 谷 博文

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