Cytosolic phospholipase A2-beta enzymes
View Patent ↗The invention provides a novel calcium-independent cytosolic phospholipase A 2 -Beta enzyme, polynucleotides encoding such enzyme and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.
1. A method for identifying an inhibitor of phospholipase activity, said method comprising:
a) contacting a phospholipid with a candidate inhibitor compound and a phospholipase enzyme peptide, wherein the phospholipase enzyme peptide comprises an amino acid sequence selected from:
i) the amino acid sequence of SEQ ID NO: 2;
ii) a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate;
iii) the amino acid sequence of SEQ ID NO: 4; and
iv) a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate; and
b) determining whether the phospholipase enzyme peptide cleaves the phospholipid and releases fatty acid, thereby identifying an inhibitor of phospholipase activity.
2. The method of claim 1 , wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO: 2.
3. The method of claim 1 , wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate.
4. The method of claim 1 , wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO: 4.
5. The method of claim 1 , wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate.
6. A method for identifying an inhibitor of phospholipase activity, said method comprising:
a) contacting a phospholipid with a candidate inhibitor compound and a phospholipase enzyme peptide, wherein the phospholipase enzyme peptide comprises an amino acid sequence selected from:
i) the amino acid sequence of SEQ ID NO: 2;
ii) a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay wish 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate;
iii) the amino acid sequence of SEQ ID NO: 4; and
iv) a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate; and
b) determining whether the phospholipase enzyme peptide activity is inhibited by the candidate inhibitor compound,
thereby identifying an inhibitor of phospholipase activity.
7. The method of claim 6 , wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO: 2.
8. The method of claim 6 , wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate.
9. The method of claim 6 , wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO: 4.
10. The method of claim 6 , wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2[ 14 C]-arachidonyl-phosphatidylcholine as a substrate.
11. A method for identifying an inhibitor of phospholipase activity, said method comprising:
a) contacting a phospholipid with a candidate inhibitor compound and a phospholipase enzyme peptide, wherein the phospholipase enzyme peptide comprises an amino acid sequence encoded by a polynucleotide that hybridizes to the nucleic acid sequence set forth in SEQ ID NO: 1 under stringent conditions of 4×SSC at 65 degrees C.; and
b) determining whether the phospholipase enzyme peptide activity is inhibited by the candidate inhibitor,
thereby identifying an inhibitor of phospholipase activity.
12. A method for identifying an inhibitor of phospholipase activity, said method comprising:
a) contacting a phospholipid with a candidate inhibitor compound and a phospholipase enzyme peptide, wherein the phospholipase enzyme peptide comprises an amino acid sequence encoded by a polynucleotide that hybridizes to the nucleic acid sequence set forth in SEQ ID NO: 3 under stringent conditions of 4×SSC at 65 degrees C.; and
b) determining whether the phospholipase enzyme peptide activity is inhibited by the candidate inhibitor,
thereby identifying an inhibitor of phospholipase activity.
13. The method of any one of claims 1 - 12 , further comprising forming a composition comprising the inhibitor and a pharmaceutically acceptable carrier.