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 eth anol and 50% ethanol-water extraction of mitochondria from beef heart or perfused rat liver, the extract was passed through thiopropyl-Sepha rose 6B column, and absorbed components were eluted with 2-mercaptoeth anol, followed by gel-filtration on Sephadex G-15. The last fraction e luted (M, about 2000) was then subjected to reverse-phase high-perform ance liquid chromatography. Of the more than 10 distinct peaks, only o ne showed specific Ca2+-channel activity in BLM with properties simila r to earlier, less extensively purified preparations, i.e., conductanc e 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 w as associated was found to have high homology with human acid alpha(1) -glycoprotein (orosomucoid) and to show identity with beef plasma oros omucoid in the Ouchterlony immunodiffusion test.