Purification of the channel component of the mitochondrial calcium uniporter and its reconstitution into planar lipid bilayers.

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 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.

journal_name

J Bioenerg Biomembr

authors

Mironova GD,Baumann M,Kolomytkin O,Krasichkova Z,Berdimuratov A,Sirota T,Virtanen I,Saris NE

doi

10.1007/BF00763072

subject

Has Abstract

pub_date

1994-04-01 00:00:00

pages

231-8

issue

2

eissn

0145-479X

issn

1573-6881

journal_volume

26

pub_type

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