O. Hayes Griffith

Emeritus Professor of Chemistry

B.A., University of California;
Ph.D., California Institute of Technology

Member of:
• Institute of Molecular Biology

Research Interests

Second messengers play a key role in transferring information from cell surface receptors. We are interested in phospholipases that release second messengers in eukaryotic cells, and also simpler isozymes produced by bacteria. Our current work is focused on phosphatidylinositol (PI) specific phospholipase C (PI-PLC). These enzymes are found in essentially all organisms. The bacterial enzymes are secreted while those of other organisms are intracellular enzymes. PI-PLCs catalyze the cleavage of the membrane lipid phosphatidylinositol (PI), or its phosphorylated derivatives, to produce diacylglycerol (DAG) and the water-soluble head group, phosphorylated myo-inositol (see diagram). The smallest PI-PLCs, about 35 kDa in size, are produced by a variety of bacteria: Bacillus cereus is a ubiquitous organism and a causative agent in both food poisoning and in some nongastrointestinal infections, e.g. meningitis in immunocompromised children. Bacillus thuringiensis has insecticide activity, and is economically important in large scale spraying of forests, orchards and crops. Bacillus anthracis causes anthrax, which results in three forms of human disease: cutaneous, gastrointestinal, and pulmonary (by inhalation of spores). Listeria monocytogenes is a food-borne pathogen that causes sepsis and meningitis in immunocompromised hosts and a maternal/fetal infection with a high mortality rate in pregnant women.

Staphylococcus aureus causes toxic shock syndrome, sepsis, colonization of joints and bones, eye and skin infections and septic arthritis. The PI-PLCs are not the primary virulence agents in these bacteria, but they are virulence factors with more subtle roles. These bacterial PI-PLCs are also important tools in membrane research due to the fact that they have the ability to release proteins tethered to membranes by a GPI (glycosylphosphatidylinositol) anchor. In eukaryotic cells, PI-PLCs play an important role in signal transduction. The binding of many extracellular signaling molecules to their cell surface receptors activates these Ca2+-dependent intracellular enzymes.

Eukaryotic PI-PLCs preferentially catalyze the hydrolysis of the phosphorylated derivative PI(4,5)P2 to generate two second messengers, diacylglycerol (DAG) and I(1,4,5)P3. The catalytic domain structure of the mammalian PI-PLC is essentially superimposable on that of the B. cereus PI-PLC despite low sequence homology. We study these enzymes using biophysical and molecular biological approaches. The kinetics are studied using novel water-soluble fluorogenic substrates synthesized in collaboration with the Keana research group, in order to avoid complications arising from scooting and hopping mode kinetics (see diagram). Allosteric effects are observed, even when the enzyme is monomer. The hypothesis is that two substrates bind to the PI-PLC, one at the active site and one at a subsite, enhancing activity. The hypothesis is being tested by studying the kinetics after addition of non-substrate short-chain phospholipids. Another project involves the development of new inhibitors for PI-PLCs. The goal is to map out the active site region and to determine the lipid surface modulates the enzyme activity, i.e. to provide a test of the hypothesis of the structure shown in the diagram, which was arrived at by combing the crystal structure of the enzyme with the probable arrangement at the interface, and to determine the dynamics of the interactions.

Selected Publications

Birrell G.B., T.O. Zaikova, A.V. Rukavishnikov, J.F. Keana, and O.H. Griffith (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., T.O. Zaikova, J.F.W. Keana, H. Goldfine, and O.H. Griffith (2002) Listeria monocytogenes phosphatidylinositol-specific phospholipase C: Activation and allostery. Biophys Chem 101-02:347-58.

Ryan M., T. Liu, F.W. Dahlquist, and O.H. Griffith (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.

Hedberg, K.K., E.B. Cogan, G.B. Birrell, and O.H. Griffith (2001) Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography. J Chromatogr B Biomed Sci Appl 757:317-24.

Zaikova, T.O., A.V. Rukavishnikov, G.B. Birrell, O.H. Griffith, and J.F.W. Keana (2001) Synthesis of Fluorogenic Substrates for Continuous Assay of Phosphatidylinositol-Specific Phospholipase C. Bioconjugate Chem 12:307-13.

Hedberg, K.K., C. Stauff, G. Hpyer-Hansen, E. Rpnne, and O.H. Griffith (2000) High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the Urokinase receptor in human glioma cells. Experimental Cell Research 257:67-81.

Griffith, O.H., and M. Ryan (1999) Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. In: Lipids, Special Thematic Issue on "Lipids in the Center," Biochem Biophys Acta 1441:237-54.

Rukavishnikov, A.V., T.O. Zaikova, G.B. Birrell, J.F.W. Keana, and O.H. Griffith (1999) Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C Bioorg Med Chem Lett 9:1133-6.

Cogan, E., G.B. Birrell, and O.H. Griffith (1999) A robotics-based automated assay for inorganic phosphate and phosphate esters. Anal Biochem 271:29-35.

Rukavishnikov, A.V., M.P. Smith, G.B. Birrell, J.F.W. Keanna, and O.H. Griffith (1998) Synthesis of a new fluorogenic substrate for the assay of phosphoinositide-specific phospholipase C Tet Lett 39:6637-40.

Liu, T., M. Ryan, F.W. Dahlquist, and O.H. Griffith (1998) Characterization of the histidine residues of B. cereus phosphatidylinositol-specific phospholipase C by NMR. In: ACS Symposium Series 718 (K.S. Bruzik, Ed., American Chemiscal Society, Washington, DC) 91-108.

Heinz, D.W., J. Wehland, and O.H. Griffith (1998) Structure and mechanisms of Ca2+ - independent phosphatidylinositol-specific phospholipase C. In: ACS Symposium Series 718 (K.S. Bruzik, Ed., American Chemiscal Society, Washington, DC) 80-90.

Rempfer, G.F., M.S. Fyfield, and O.H. Griffith (1998) Lenses for electron microscopy and microanalysis: the shadowgraph method of determining focal properties and aberration coefficients. Microscopy and Microanalysis 4:34-49.

Rukavishnikov, A.V., M. Ryan, O.H. Griffith, and J.F.W. Keana (1997) A chromogenic substrate for the continuous assay of mammalian phosphatidylinositol-specific phospholipase C Bioorganic and Medicinal Chem Lett 7:1239-42.

Birrell, G..B., K.K. Hedberg, E. Barklis, and O.H. Griffith (1997) Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C J Cellular Biochem 65:550-4.

Rempfer, G.F., D.M. Desloge, W.P. Skoczylas, and O.H. Griffith (1997) Simultaneous correction of spherical and chromatic aberrations with an electron mirror: An electron optical achromat. Microscopy and Microanalysis 3:14-27.

Heinz, D.W., M. Ryan, M.P. Smith, L.H. Weaver, J.F.W. Keana, and O.H. Griffith (1996) Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl (1,6)-D-myo-inositol, an essential fragment of GPI anchors. Biochemistry 35:9496-504.

Ryan, M., M.P. Smith, T.K. Vinod, W.L. Lau, J.F.W. Keana, and O.H. Griffith (1996) Synthesis, structure-activity relationships,and the effect of polyethylene glycol on inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. J Med Chem 39:4366-76.

Habliston, D.L., K.K. Hedberg, G.B. Birrell, G.F. Rempfer, and O.H. Griffith (1995) Photoelectron imaging of cells: Photoconductivity extends the range of applicability. Biophys J 69:1615-24.

Heinz, D.W., M. Ryan, T.L. Bullock, and O.H. Griffith (1995) Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol. EMBO J 14:3855-63.

Birrell, G.B., K.K. Hedberg, and O.H. Griffith (1995) An extracellular phospholipid-specific phospholipase C is released by cultured Swiss 3T3 cells. Biochem Biophys Res Comm 211:318-24.

Kaneko, K., D. Peretz, K.K. Pan, T.C. Blochberger, H. Wille, R. Gabizon, O.H. Griffith, F.E. Cohen, M.A. Baldwin, and S.B. Prusiner (1995) Prion protein (Pr) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform. PNAS 92:11160-4.

Hedberg, K.K., G.B. Birrell, D.L. Habliston, and O.H. Griffith. (1995) Tunable label contrast on the cell surface: photoelectron imaging with multiple wavelength excitation. J. Microscopy Soc. of Amer. 1:253-61.

Hedberg, K.K., G.B. Birrell, P.L. Mobley, and O.H. Griffith. (1994) Transition metal chelator TPEN counteracts phorbol ester-induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C. J. Cell. Phys. 158:337-46.

Mobley, P.L., K.K. Hedberg, O.H. Griffith, L. Rochat, and B. Bhen. (1994) Decreased phosphorylation of four 20 kDa proteins precedes staurosporine-induced disruption of the actin-myosin cytoskeleton in rat astrocytes. Exper. Cell. Research 214:55-66.

Ryan, M., J.C. Huang, O.H. Griffith, J.F.W. Keana, and J.J. Volwerk. (1994) A chemiluminescent substrate for the detection of phosphatidylinositol-specific phospholipase C. Anal. Biochem. 214:548-56.

Birrell, G.B., K.K. Hedberg, J.J. Volwerk, and O.H. Griffith. (1993) Differential expression of phospholipase C specific for inositol phospholipids at the cell surface of rat glial cells and REF52 rat embryo fibroblasts. J. Neurochem. 60:620-5.

Habliston, D.L., G.B. Birrell, O.H. Griffith, and G.F. Rempfer. (1993) Photoelectron imaging of DNA: A study of substrates and contrast. J. Phys. Chem. 97:3022-7.

Bullock, T.L., M. Ryan, S.L. Kim, S.J. Remington, and O.H. Griffith. (1993) Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus. Biophys. J 64:784-91.

Griffith, O.H., K.K. Hedberg, D. Desloge, and G.F. Rempfer. (1992) Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system. J. Microscopy 168:249-58.

Leigh, A., J.J. Volwerk, O.H Griffith, and J.F.W. Keana. (1992) The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic 4-Nitrophenyl i-inositol-1-phosphate. Biochemistry 31:8978-83.