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).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025698-30
Application #
2021801
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1978-06-01
Project End
1998-02-28
Budget Start
1996-12-01
Budget End
1998-02-28
Support Year
30
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Birrell, G Bruce; Zaikova, Tatiana O; Rukavishnikov, Aleksey V et al. (2003) Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C. Biophys J 84:3264-75
Ryan, M; Liu, T; Dahlquist, F W et al. (2001) A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C. Biochemistry 40:9743-50
Zaikova, T O; Rukavishnikov, A V; Birrell, G B et al. (2001) Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug Chem 12:307-13
Hedberg, K K; Stauff, C; Hoyer-Hansen, G et al. (2000) High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the urokinase receptor in human glioma cells. Exp Cell Res 257:67-81
Griffith, O H; Ryan, M (1999) Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. Biochim Biophys Acta 1441:237-54
Rukavishnikov, A V; Zaikova, T O; Birrell, G B et al. (1999) Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C. Bioorg Med Chem Lett 9:1133-6
Menco, B P; Birrell, G B; Fuller, C M et al. (1998) Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface. Chem Senses 23:137-49
Liu, T; Ryan, M; Dahlquist, F W et al. (1997) Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis. Protein Sci 6:1937-44
Gassler, C S; Ryan, M; Liu, T et al. (1997) Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis. Biochemistry 36:12802-13
Birrell, G B; Hedberg, K K; Barklis, E et al. (1997) Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C. J Cell Biochem 65:550-64

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