Phospholipase assays

IR spectroscopy of the X-receptor protein signal peptide in phospholipid monolayers shows that the peptide affects the packing of the lipid hydrocarbon tails (M. S. Briggs, R. A. Dluhy, D. G. Cornell, and L. M. Gierasch, unpublished results). In samples formed at the same surface pressure, the lipid tails are oriented differendy in the presence and absence of signal peptide. A phospholipase assay for structural defects in phospholipid bilayers (Jain et al., 1984) indicates that the X-receptor protein signal peptide interacts with vesicles to induce such defects. The peptides perturb the lipid structure at lower mole fractions than do various lysophospholipids. These data provide yet another indication that signal peptides interact with and perturb lipid complexes. 

The subject of phospholipase assays has been concisely reviewed [1-5]. A standard approach employs phospholipids with radioisotopes incorporated into specific positions in the molecule. By the appropriate choice of labeling, the specificity of the enzymes can readily be established and as little as a few picomoles of product can be detected. The use of isotopes has been helpful in the measurement of phospholipase activity using the membranes of whole cells or isolated subcellular fractions previously labeled with radioactive phospholipid precursors. 

However, extreme care must be exercised when trying to draw physiological conclusions from studies using nonphysiological substrates (i.e., p-nitrophenyl esters or 4-methylumbelliferyl esters). We are currently investigating a new phospholipase assay (28), which employs a fluorescent phospholipid substrate, l-acyl-2-l6-((7-nitro-2,1,3 benzoxadiazol-4 yl)amino]-caproyl] phosphatidylcholine (Ca-NBD-PC). Our preliminary studies (Table IV) indicate... 

Another popular assay for GPCR activation is to measure the increase in intracellular Ca2+ that occurs upon activation. GPCRs on the cell surface produce inositol triphosphate (IP3) via the action of Phospholipase C (PLC). IP3 stimulates calcium channels called IP3 receptors on the endoplasmic reticulum, which raise... 

Hendrickson, H. S., Hendrickson, E. K., Johnson, I. D. and Farber, S. A. (1999). Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging. Anal. Biochem. 276, 27-35. 

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

Ghosh, M. and Smrcka, A. V. Assay for G protein-dependent activation of phospholipase C beta using purified protein components. Methods Mol. Biol. 237 67-75, 2004. 

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

A colorimetric assay for lecithin and choline was described by Kotsira and Klonis (1998) using two enzymes (phospholipase and choline oxidase) and an indicator dye conjugate (bromothymol blue-glutathione) co-immobilised on a glutaraldehyde-activated polyacrylamide transparent gel. The change of the... 

Selected entries from Methods in Enzymology [vol, page(s)] Cobra venom phospholipase A2 Naja naja naja, 197, 359 phospholipase A2 from rat liver mitochondria, 197, 365 assay and purification of phospholipase A2 from human synovial fluid in rheumatoid arthritis, 197, 373 purification of mammalian nonpan-creatic extracellular phospholipases A2, 197, 381 spleen phospholipases A2, 197, 390 purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets, 197, 400. 

Selected entries from Methods in Enzymology [vol, page(s)] Phosphatidylinositol-specific phospholipases C from Bacillus ce-reus and Bacillus thuringiensis, 197, 493 assays for phosphoinosi-tide-specific phospholipase C and purification of isozymes from bovine brain, 197, 502 properties of phospholipase C isozymes, 197, 511 phosphatidylinositol-specific phospholipase C from human platelets, 197, 518 purification of guinea pig uterus phos-phoinositide-specific phospholipase C, 197, 526. 

Lowe, M.E. 1999. Assays for pancreatic triglyceride lipase and colipase. In Methods in Molecular Biology, Vol. 109 Lipase and Phospholipase Protocols (M.H. Doolittle and K. Reue, eds.) pp. 59-70. Humana Press, Totowa, N.J. 

This book provides a complete coverage of phospholipases from the technicalities of assay of phospholipases to the proposed mechanism of catalysis. 

Kinkaid, A. and Wilton, D.C. 1991. Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay. Biochem. J. 278, 843-848. 

Hanna PA, Jankovic J, Vincent A (1999) Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies. J Neurol Neurosurg Psychiatry 66 612-16 Hanson MA, Stevens RC (2000) Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution. Nat Struct Biol 7 687-92 Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog s neuromuscular junction. Nature 409 479-84 Harris JB (1997) Toxic phospholipases in snake venom an introductory review. Symp. zool. Soc. Lond. 70 235-50... 

Having shown that dibutyryl PC is monomeric under the enzyme assay conditions, we found that the phospholipase A2, which acts poorly on PE in mixed micelles, is activated by dibutyryl PC which is itself an even poorer substrate. 31p-NMR spectroscopy was employed to show that only PE is hydrolyzed in mixtures of various compositions of these two phospholipids. The fully activated enzyme hydrolyzes PE at a similar rate to its optimal substrate, PC containing long-chain fatty acid groups. Because dibutyryl PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzyme by phosphoryl-choline-containing lipids (either monomeric or micellar) rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Therefore, two functional sites on the enzyme have to be assumed an activator site and a catalytic site (6). 

Design and Development of a Selective Assay System for the Phospholipase A2 Superfamily... 

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