Characterization of phospholipase A2 activity in reticulocyte endocytic vesicles.
Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A2 activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spin-labeled phospholipid analogues, we measured the hydrolysis rate of the ester bond at position 2 during incubation with reticulocyte endocytic vesicles. We have shown that this phospholipase A2 activity was membrane-associated, enriched in endocytic vesicles as compared to cytosol and plasma membrane. Enzymic activity was also observed in exosomes, vesicles coming from the endocytic compartment and released by reticulocytes during their maturation in erythrocytes. Neither the hydrolytic activity nor the membrane association was found to be Ca(2+)-dependent. Spin-labeled phospholipids with choline and serine polar heads were better substrates than glycerophosphoethanolamine analogues. Optimal pH was found to be close to neutral. 5,5'-Dithiobis(2-nitrobenzoic acid) and diisopropyl fluorophosphate very efficiently inhibited spin-labeled phospholipid hydrolysis. This phospholipase A2 activity was confirmed using a radioactive assay, although with much lower sensitivity. (E)-6-(Bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a specific mechanism-based inhibitor of calcium-independent phospholipases A2, was found to abolish the enzymic activity present in endocytic vesicles.
Bette-Bobillo
P
P
URA 1856 CNRS, Université Montpellier II, France.
Vidal
M
M
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000201:enzymology* / Q000648:ultrastructure
D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium