Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane.
A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002463:Cell Membrane Permeability; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000032:analysis; D008565:Membrane Proteins / Q000302:isolation & purification*; D008930:Mitochondria, Liver / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D018272:Porins; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D008565:Membrane Proteins; D010455:Peptides; D018272:Porins