Batten disease : HPLC separation of the c subunits of mitochondrial and vacuolar ATPase stored in Batten disease

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of fatal inherited neurodegenerative diseases of children and animals, characterised by retinal and brain atrophy. A common finding in these diseases is the occurrence of intracellular fluorescent storage bodies, which contain the hydrophobic c subunit of mitochondrial ATP synthase. The accumulation of the analogous vacuolar ATPase c subunit has also been noted in tissues in some forms of the disease. Pure storage body preparations were obtained from liver and pancreas from affected sheep and liver from affected Border Collie dogs. Analysis of the isolated storage bodies by polyacrylamide gel electrophoresis revealed significant amounts of vacuolar ATPase subunit c, particularly in affected Border Collie liver preparations. This established that the vacuolar ATPase c subunit is a genuine storage body component and does not arise from co-purification of vacuolar membrane. The c subunits are hydrophobic proteins which have the tendency to irreversibly aggregate and do not dissolve in aqueous solvents. They cannot be separated by aqueous solvent based chromatographic techniques. High performance liquid chromatography techniques using normal phase silica chromatography and DEAE-cellulose ion exchange chromatography and chloroform:methanol:water based solvents were developed. The stored vacuolar ATPase subunit c was separated from the mitochondrial ATP synthase c subunit by these methods. Lack of reproducibility was a common occurrence with chromatograms of similar samples dramatically changing over a period of days. Problems with reproducibility were ascribed to the formation of oligomers, oxidation, and to irreversible precipitation of the hydrophobic proteins onto the column. The addition of trifluoroacetic acid and ammonium acetate to the solvents partially resolved these problems. Addition of mercaptoethanol, an antioxidant, unexpectedly resulted in a greater lack of reproducibility. These findings lead to the conclusion that high performance liquid chromatographic techniques are not suitable for repetitive assays of the relative proportions of the two c subunits in storage bodies.