|ZFIN ID: ZDB-PUB-000103-5|
Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging
Hendrickson, H.S., Hendrickson, E.K., Johnson, I.D., and Farber, S.A.
|Source:||Analytical biochemistry 276(1): 27-35 (Journal)|
|Registered Authors:||Farber, Steven|
|Keywords:||BODIPY; cytosolic (85 kDa) phospholipase A(2); plasma platelet-activating factor; acetylhydrolase; fluorescence; quenching; zebrafish; embryo; imaging; assay|
|PubMed:||10585741 Full text @ Anal. Biochem.|
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. Analytical biochemistry. 276(1):27-35.
ABSTRACTPhospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A(2) (cPLA(2)) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA(2) (specific activities: 18 and 5 nmol min(-1) mg(-1)) and all were good for rPAF-AH (specific activities: 17, 11, and 6 μmol min(-1) mg(-1)). The minimal amount of enzyme detectable was 50 ng for cPLA(2) and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA(2) in zebrafish embryo extracts and one was well suited for imaging of PLA(2) activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA(2) activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.
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