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Structure, evolution and regulation of venom and mammalian secreted PLA2's

Florence Davidson

Appointment Period: 1985-1989, Grant Years: [01,02,03,04]

The effects of the proposed PLA2-regulatory proteins lipocortins I and II were investigated in the assay systems with which they were initially identified; i.e., using the porcine pancreatic phospholipase A2 and E. coli, PC(phosphorylcholine)/deoxycholate mixtures, or PC liposomes as substrate. The Naja naja naja PLA2 and a PLA2 from a murine macrophage-like cell line were also tested. It was found that inhibition was only observed at very low substrate concentrations in all of these systems, and the purified lipocortins could bind to substrates and cofactor directly. Inhibition could be overcome by raising the substrate concentration, or by using a different substrate. In the E. coli system, the inhibition occurs by depletion of substrate, rather than direct effects on the enzyme. In the PC/deoxycholate system, inhibition is probably due to depletion of the essential cofactor Ca2. With PC vesicles as a substrate, the results are consistent with inhibition by substrate depletion, but competitive inhibition involving a rather weak KI cannot be ruled out. The in vitro studies do not yet support a proposal that these proteins specifically inhibit PLA2's in vivo.

The effects of the substrate analog 1-stearyl,2-stearoylaminodeoxy phosphatidylcholine on the hydrolysis of various substrates by the Naja naja naja PLA2 were also examined. The compound is a potent reversible inhibitor when PC is used as substrate, but an activator of PE hydrolysis by the PLA2. Such analogs should prove useful for investigating the dual phospholipid model of activation of the PLA2.

PUBLICATIONS (resulting from this training, and some more recent ones)

Pluckthun A, Rohlfs R, Davidson FF, Dennis EA. (1985) Short-chain phosphatidylethanolamines: physical properties and susceptibility of the monomers to phospholipase A2 action. Biochemistry. 24:4201-8.

Davidson FF, Hajdu J, Dennis EA. (1986) 1-Stearyl,2-stearoylaminodeoxy phosphatidylcholine, a potent reversible inhibitor of phospholipase A2. Biochem Biophys Res Commun. 137:587-92.

Davidson FF, Dennis EA, Powell M, Glenney JR Jr. (1987) Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids. J Biol Chem. 262:1698-705.

Davidson FF, Dennis EA. (1989) Biological relevance of lipocortins and related proteins as inhibitors of phospholipase A2. Biochem Pharmacol. 38:3645-51.

Dennis EA, Davidson FF. (1990) Phospholipase A2 and lipocortin effects. Prog Clin Biol Res. 349:47-54.

Davidson FF, Dennis EA. (1990) Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2. Biochim Biophys Acta. 1037:7-15.

Davidson FF, Lister MD, Dennis EA. (1990) Binding and inhibition studies on lipocortins using phosphatidylcholine vesicles and phospholipase A2 from snake venom, pancreas, and a macrophage-like cell line. J Biol Chem. 265:5602-9.

Davidson FF, Dennis EA. (1990) Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms. J Mol Evol. 31:228-38.

Davidson FF, Dennis EA. (1992) Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins. Biochim Biophys Acta. 1127:270-6.

Davidson FF, Loewen PC, Khorana HG. (1994) Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. Proc Natl Acad Sci U S A. 91:4029-33.

Mollaaghababa R, Davidson FF, Kaiser C, Khorana HG. (1996) Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11482-6. Erratum in: Proc Natl Acad Sci U S A 94:3481.

Davidson FF, Steller H. (1998) Blocking apoptosis prevents blindness in Drosophila retinal degeneration mutants. Nature. 391:587-91.