Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.
A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.
Hagopian
K
K
Department of Neurochemistry, Institute of Neurology, Queen Square, London, WC1N 3BG, United Kingdom. khagopian@facstaff.wisc.edu
D000818:Animals; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D009472:Neuronal Ceroid-Lipofuscinoses / Q000201:enzymology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D012995:Solubility; D013367:Submitochondrial Particles / Q000201:enzymology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D003902:Detergents; D014361:Trypsin Inhibitors; D012967:Sodium Dodecyl Sulfate; C028913:dodecyl sulfate; D002918:Chymotrypsin; D006180:Proton-Translocating ATPases