The central them of this research program is lipid-protein interaction involved in the function of membrane enzymes and signal transduction. The four main sub-areas are: a) Cardiolipin (diphosphatidylglycerol, DPG) association with cytochrome c oxidase.
The aim i s to determine the location and binding constants of the 2 s molecules of DPG that co-purify with this mitochondrial enzyme complex. The methodology involves a newly synthesized DPG analog that will covalently link to polypeptides of cytochrome c oxidase and a spin-labeled DPG to determine the relative binding constants of this unusual lipid. b) Mapping of the lipid binding region of phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC cleaves and PI into two parts: lipid-soluble diacylglycerol (DG) and water-soluble inositol phosphate. The bacterial enzyme is implicated in the release of membrane proteins anchored by PI. In higher eukaryotes PI-PLC acts as a biological amplifier and produces DG and inositol triphosphate (IP3), both of which are secondary messengers in the PI signal transduction pathway. IP-PLC from Bacillus cereus and other bacteria, will be studied by initial rate kinetics, covalent lipid labeling, ad spin-labeling. The questions being asked involve whether PI-PLC has an extended lipid binding region as proposed for other lipolytic enzyme, the nature of the interactions between the protein and the lipid substrate, and which amino acid residues play a role in this interaction. c) New affinity ligands for the isolation of PI-specific phospholipase c. The goal of this effort is to overcome the difficulties in isolation of bacterial and mammalian PI-PLC. The affinity matrix will be tested on the bacterial enzymes and then used to isolated mammalian PI-PLCs involved in the signal transduction pathway. d) Biophysical studies of lipid and cofactor binding to mammalian protein kinase c (PKC). The DG generated by PI PLC in higher eukaryotes activates this key enzyme of the PI signal transduction pathway. PKC will be examined by a combination of spin labeling and biochemical methods to address questions involving the penetration of PKC into the phospholipid bilayer after the translocation step, the relative binding parameters of the secondary messenger DG and the phorbol ester tumor prometers, and the location of the ATP binding site. The long range goal of aims b-d is to provide a sound biochemical and biophysical understanding of molecular events involved in signal transduction. This pathway is linked to the action of certain growth factors and oncogenes. Subversions of it are implicated in cancer and several genetic disorders (eg. Krabbes and Gaucher's diseases).
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