Abstract
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 r 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 20 pS and multiples thereof, clustering of channels, participation of 2 or more subunits in channel formation, and sensitivity to 1 µM 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 α1-glycoprotein (orosomucoid) and to show identity with beef plasma orosomucoid in the Ouchterlony immunodiffusion test.
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Mironova, G.D., Baumann, M., Kolomytkin, O. et al. Purification of the channel component of the mitochondrial calcium uniporter and its reconstitution into planar lipid bilayers. J Bioenerg Biomembr 26, 231–238 (1994). https://doi.org/10.1007/BF00763072
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DOI: https://doi.org/10.1007/BF00763072