USE OF ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY AND TANDEM MASS-SPECTROMETRY TO STUDY BINDING OF F-0 INHIBITORS TO CEROID-LIPOFUSCINOSIS PROTEIN, A MODEL SYSTEM FOR SUBUNIT-C OF MITOCHONDRIAL ATP SYNTHASE

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence th at is identical to that of the F-0 subunit c of normal bovine ATP synt hase, Electrospray ionization mass spectrometry (ESI-MS) has shown tha t ovine CLP and normal bovine F-0 subunit c are identical, including a 42 mass unit post-translational modification, Although the identity a nd the location of this modification have not been fully established i n both species, CLP can be used as a convenient and a unique source of subunit c for studies of F-0 inhibitor interactions by ESI-MS analysi s. Analysis of mixtures of CLP incubated with several known F-0 inhibi tors showed that N,N'-dicyclohexyl-carbodiimide and organotins bind co valently to CLP but interactions with oligomycin and venturicidin were not observed, The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,- benzoquinone (UQ(0)) and N-ethyl maleimide (NEM) were also shown to bi nd covalently to the protein, The binding stoichiometry and the relati ve rate of reaction were then determined for each inhibitor. Tandem ma ss spectrometry experiments performed on the [M+5H](5+) ion of the int act CLP and of the complexes UQ(0)-CLP and NEM-CLP snowed the identifi cation of 80% of the CLP sequence and revealed that UQ(0) and NEM are both bound to cysteine-64, This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F-0 component of ATP synthase.