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(r) about 2000) was then subjected to reverse-phase high-performance liquid chromatography. Of the more than 10 distinct peaks, only one showed specific Ca(2+)-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 microM ruthenium red. Voltage sensitivity and cooperativity between channels are described. The Ca(2+)-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.
Mironova
G D
GD
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino.
Baumann
M
M
Kolomytkin
O
O
Krasichkova
Z
Z
Berdimuratov
A
A
Sirota
T
T
Virtanen
I
I
Saris
N E
NE
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000235:genetics / Q000276:immunology; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D009961:Orosomucoid; D010455:Peptides; C034703:mitochondrial calcium uniporter