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TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 599 |
Fluorogenic
Phospholipids as Head Group-Selective Reporters of Phospholipase A Activity |
Tyler
M. Rose and Glenn D. Prestwich |
phospholipases
A, cell signaling, lisophospholipids, PLA2 enzymes |
Dissociated neurons from 8–16
week-old SCG were plated at a density of one-half ganglion per one 35 mm poly-l-lysine-coated
glass bottom petri dish (no. 1.5, MatTek Corp.) and incubated at 37 °C in a 5% CO2 environment
in MEM.
|
video-rate
confocal microscopy |
mouse |
neuronal |
SCG
neurons |
| Abstract |
| |
PLA (phospholipases A) are important mediators of
cell signaling, generating bioactive fatty acids and LPLs (lysophospholipids). PLA products
having different head groups can initiate vastly different types of signaling. Fluorogenic analogues
of the PLs (phospholipids) PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine),
and PG (phosphatidylglycerol) were synthesized as PLA substrates for rapidly determining in
real time the influence of head group modifications on cell signaling both in vitro and in cells.
Enzymeassisted remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom
PLA2 and transphosphatidylation with a particular PLD (phospholipase D) were central steps in
the preparation of these enzymatic probes. The resulting fluorogenic Dabcyl- and BODIPY-containing
PL analogues, DBPA, DBPC, DBPE, and DBPG, were used in mixed micelle assays to determine PLA2
kinetics. Next, the assays were used to determine the Xi(50) value of a common PLA2 inhibitor.
Finally, the head group selectivities of a series of commercially available PLA2 enzymes were
readily established using the DBPLs (Dabcyl-BODIPY PLs) as substrates. |
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