CHARACTERIZATION OF PHOSPHOLIPASE A(2) ACTIVITY IN RETICULOCYTE ENDOCYTIC VESICLES

Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A(2) activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spinlabeled 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 A(2) 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 Ca2+-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 A(2) 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 A(2), was found to abolish the enzymic activity present in endocytic vesicles.