alpha-chymotrypsin and Neuronal-Ceroid-Lipofuscinoses

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.

Topics: Animals; Chromatography, Gel; Chymotrypsin; Detergents; Electrophoresis, Polyacrylamide Gel; Humans; Male; Mitochondria, Liver; Molecular Weight; Neuronal Ceroid-Lipofuscinoses; Protein Conformation; Proton-Translocating ATPases; Rats; Rats, Wistar; Sodium Dodecyl Sulfate; Solubility; Submitochondrial Particles; Trypsin Inhibitors