PURIFICATION OF A K+-CHANNEL PROTEIN OF SARCOPLASMIC-RETICULUM BY ASSAYING THE CHANNEL ACTIVITY IN THE PLANAR LIPID BILAYER SYSTEM

被引：15

作者：

IDE, T ^{[1
]}

MORITA, T ^{[1
]}

KAWASAKI, T ^{[1
]}

TAGUCHI, T ^{[1
]}

KASAI, M ^{[1
]}

机构：

[1] OSAKA UNIV,FAC ENGN SCI,DEPT BIOPHYS ENGN,TOYONAKA,OSAKA 560,JAPAN

来源：

BIOCHIMICA ET BIOPHYSICA ACTA | 1991年 / 1067卷 / 02期

关键词：

POTASSIUM ION CHANNEL; SARCOPLASMIC RETICULUM; RECONSTITUTED LIPID VESICLE; LIPID BILAYER METHOD;

D O I：

10.1016/0005-2736(91)90046-B

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A K+ channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K+ channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca2+-ATPase antibody did not recognize the protein. The characteristics of the K+ channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCI and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.

引用

页码：213 / 220

页数：8

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