Intramolecularly Quenched BODIPY-Labeled Phospholipid Analogs in Phospholipase A2 and Platelet-Activating Factor Acetylhydrolase Assays and in Vivo Fluorescence Imaging
✍ Scribed by H.Stewart Hendrickson; Elizabeth K. Hendrickson; Iain D. Johnson; Steven A. Farber
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 200 KB
- Volume
- 276
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Phospholipase 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 mol min ؊1 mg ؊1 ). The minimal amount of enzyme de- tectable 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.