ElectrosDrav Ionisation Mass SDectrometrv (ESVMS) of Ceroid LiDOfuscin Protein : A Model Svstem for the Study of FO Inhibitor Interactions with Mitochondria1 Subunit C

Ceroid lipofuscin protein (CLP) accumuiates in lysosome derived storage bodies in a number of different forms of the inherited neuro-degenerative diseases, the neuronal ceroidlipofuscinoses, also called Batten Disease. It is derived from genes coding for subunit C, a subunit of the FO component of mitochondrial ATP synthase. The gene sequence gives the same amino acid sequence in rats, sheep, cattle and humans. The sequence predicts a 75 amino acid protein of mass 7608 Da (1,2). CLP was extracted in high yield and purity from storage bodies from affected sheep by chloroform/methanol extraction ( I ) . In contrast, chloroform/methanol extraction of mitochondria gives subunit C in a low yield and other proteins are co-extracted (3). CLP thus has significant advantages for studies of FOinhibitor interactions and provides a model system for mitochondrial subunit C interactions by ESIMS. ESI/MS has not been widely used on hydrophobic proteins because they are not soluble in the solvent systems most commonly used (acetonitrile/water, methanol/water) and most detergents used to solubilize hydrophobic proteins interfere with the assay. However, recent studies (4) have shown that chloroformhethanol mixtures are suitable solvents for hydrophobic proteins and these have been used in the present study.