PURIFICATION OF THE CHANNEL COMPONENT OF THE MITOCHONDRIAL CALCIUM UNIPORTER AND ITS RECONSTITUTION INTO PLANAR LIPID BILAYERS

The purification of the channel-forming component of the mitochondrial calcium uniporter and its channel properties are described. After ethanol and 50% ethanol-water extraction of mitochondria from beef heart or perfused rat liver, the extract was passed through thiopropyl-Sepharose 6B column, and absorbed components were eluted with 2-mercaptoethanol, followed by gel-filtration on Sephadex G-15. The last fraction eluted (M, about 2000) was then subjected to reverse-phase high-performance liquid chromatography. Of the more than 10 distinct peaks, only one showed specific Ca2+-channel activity in BLM with properties similar to earlier, less extensively purified preparations, i.e., conductance of 20pS and multiples thereof, clustering of channels, participation of 2 or more subunits in channel formation, and sensitivity to 1 mu M ruthenium red. Voltage sensitivity and cooperativity between channels are described. The Ca2+-binding glycoprotein with which the peptide was associated was found to have high homology with human acid alpha(1)-glycoprotein (orosomucoid) and to show identity with beef plasma orosomucoid in the Ouchterlony immunodiffusion test.