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Cytosolic phospholipase A2-beta enzymes

a technology of cytosolic phospholipase and enzyme, applied in the field of cytosolic phospholipase a2beta enzyme, can solve the problems of limited effectiveness of the present anti-inflammatory drugs which act through inhibition of the arachidonic acid cascade steps, and is not believed to contribute to prostaglandin and leukotriene production. , to achieve the effect of reducing inflammation

Inactive Publication Date: 2006-01-26
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0027] The present invention also provides methods for identifying an inhibitor of phospholipase activity said method comprising: (a) combining a phospholipid, a candidate inhibitor compound, and a composition comprising a phospholipase enzyme peptide; and (b) observing whether said phospholipase enzyme peptide cleaves said phospholipid and releases fatty acid thereby, wherein...

Problems solved by technology

The efficacy of the present anti-inflammatory drugs which act through inhibition of arachidonic acid cascade steps is limited by the existence of side effects which may be harmful to various individuals.
However, because the secreted phospholipase A2 enzymes are extracellular proteins (i.e., not cytosolic) and do not selectively hydrolyze arachidonic acid, they are presently not believed to contribute to prostaglandin and leukotriene production.

Method used

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  • Cytosolic phospholipase A2-beta enzymes
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Examples

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example 1

Library Construction

[0052] Oligo-dT primed and random primed cDNA libraries were constructed from U937 cells using a Poly A Track kit for isolation of mRNA (Promega), a Superscript Choice kit for the generation of double stranded cDNA (Gibco BRL), and a Lambda ZapII phage cloning kit (Stratagene).

Clone Identification

[0053] Two cPLA2-β specific deoxyribonucleotides were designed based on the sequence of EST clone W92213:

5′-CCTCCTGCAGCCCACTCGGGAC-3′(SEQ ID NO:5)5′-GCTGACCAGAGGAAAGTGCAGC-3′(SEQ ID NQ:6)

These oligonucleotides were used to screen 106 recombinates of both the oligo dT primed and random primed library. One clone which hybridizes with both oligonucleotides, clone 52A, was examined for complete DNA sequence determination (SEQ ID NO:1). The partial coding sequence on this clone begins at nucleotide 1560 and continues to a stop codon at nucleotide 3894, representing 778 amino acids (see SEQ ID NO:2). The region on the DNA sequence 5′ to nucleotide 1560 fits a splice ac...

example 2

Phopholipase Assays

1. sn-2 Hydrolysis Assays

[0058] A) Liposome: The lipid, e.g. 1-palmitoyl-2-[14C]arachidonyl-sn-glycero-3-phosphocholine(PAPC), 55 mCi / mmol, was dried under a stream of nitrogen and solubilized in ethanol. The assay buffer contained 100 mM Tris-HCl pH 7, 4 mM EDTA, 4 mM EGTA, 10% glycerol and 25 μM of labelled PAPC, where the volume of ethanol added was no more than 10% of the final assay volume. The reaction was incubated for 30 minutes at 37° C. and quenched by the addition of two volumes of heptane:isopropanol:0.5M sulfuric acid (105:20:1 v / v). Half of the organic was applied to a disposable silica gel column in a vacuum manifold positioned over a scintillation vial, and the free arachidonic was eluted by the addition of ethyl ether (1 ml). The level of radioactivity was measured by liquid scintillation.

[0059] Variations on this assay replace EDTA and EGTA with 10 mM CaCl2.

[0060] B) Mixed Micelle Basic: The lipid was dried down as in (A) and to this was add...

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Abstract

The invention provides a novel calcium-independent cytosolic phospholipase A2-Beta enzyme, polynucleotides encoding such enzyme and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.

Description

[0001] The present invention relates to a purified cytosolic phospholipase A2-Beta (cPLA2-β) enzymes which are useful for assaying chemical agents for anti-inflammatory activity. BACKGROUND OF THE INVENTION [0002] The phospholipase A2 enzymes comprise a widely distributed family of enzymes which catalyze the hydrolysis of the acyl ester bond of glycerophospholipids at the sn-2 position. One kind of phospholipase A2 enzymes, secreted phospholipase A2 or sPLA2, are involved in a number of biological functions, including phospholipid digestion, the toxic activities of numerous venoms, and potential antibacterial activities. A second kind of phospholipase A2 enzymes, the intracellular phospholipase A2 enzymes, also known as cytosolic phospholipase A2 or cPLA2, are active in membrane phospholipid turnover and in regulation of intracellular signalling mediated by the multiple components of the well-known arachidonic acid cascade. One or more cPLA2 enzymes are believed to be responsible fo...

Claims

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Application Information

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IPC IPC(8): C12N9/20C07H21/04C12P21/06
CPCC12N9/20C07K16/40
Inventor KRIZ, RONALDSONG, CHUANZHENG
Owner WYETH LLC
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