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
✍ Scribed by Armelle Buzy; Edward M. Ryan; Keith R. Jennings; David N. Palmer; David E. Griffiths
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 664 KB
- Volume
- 10
- Category
- Article
- ISSN
- 0951-4198
No coin nor oath required. For personal study only.
✦ Synopsis
Ceroid lipofuscinosiiprotein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the Fo subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine Fo subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F, inhibitor interactions by ESI-MS analysis.
Analysis of mixtures of CLP incubated with several known F, inhibitors showed that N,N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQo) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5I@+ ion of the intact CLP and of the complexes UQo-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ, 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 Fo component of ATP synthase.