Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis.
We have developed a simple fluorescent assay for detection of phospholipase A2 (PLA2) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA2 inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca2+-dependent cytosolic PLA2 (cPLA2) subtype. Embryonic cPLA2 activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA2, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.
Farber
S A
SA
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210, USA. Farber@mail1.ciwemb.edu
Olson
E S
ES
Clark
J D
JD
Halpern
M E
ME
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001756:Blastoderm / Q000201:enzymology; D001896:Boron Compounds; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D005775:Gastrula / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D007425:Intracellular Membranes; D008297:Male; D008956:Models, Chemical; D063065:Organophosphonates / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D015027:Zebrafish / Q000196:embryology*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001095:Arachidonic Acids; D001896:Boron Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D063065:Organophosphonates; C104810:methyl arachidonylfluorophosphonate; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium