Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system.
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-Ca(2+)-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 KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.
Ide
T
T
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Japan.
Morita
T
T
Kawasaki
T
T
Taguchi
T
T
Kasai
M
M
D000818:Animals; D015153:Blotting, Western; D002793:Cholic Acids; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008051:Lipid Bilayers; D015221:Potassium Channels / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*
D002793:Cholic Acids; D008051:Lipid Bilayers; D015221:Potassium Channels; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate