Localization and partial purification of a neutral-active phospholipase A2 from BCG-induced rabbit alveolar macrophages.
The localization and partial purification of a Ca2+-dependent, membrane associated phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from BCG-induced rabbit alveolar macrophages is described. Phospholipase A activity was determined using autoclaved Escherichia coli, the phospholipids of which were labelled in the 2-acyl position with [1-14C]oleate. Sonicated macrophages or granule preparations exhibited maximal phospholipase A2 activity at pH 7.0, with 5 mM Ca2+. Activity was quantitatively recovered in the pellet after centrifugation of homogenates at 100 000 x g, indicating that the enzyme is membrane-associated. At least two populations of macrophage granules were separated that contained phospholipase A2 activity. Plasma membranes enriched 15-fold with respect to alkaline phosphodiesterase I were devoid of phospholipase activity. The enzyme was purified 1278-fold in a yield of 34%, was active over a broad pH range, and was extremely sensitive to low concentrations of Ca2+. Mg2+ and Mn2+ would not substitute for Ca2+, 1 mM EDTA completely inhibited enzymatic activity. Absolute specificity for the 2-position was demonstrated using 1-[1-14C]stearyl-2-acyl 3-sn-glycerophosphorylethanolamine as substrate. Phospholipase A2 activity was inhibited by the nonsteroidal anti-inflammatory agent indomethacin; the amount of drug required for 50% inhibition was 5 . 10(-4) M.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007213:Indomethacin / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology*; D009163:Mycobacterium bovis; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology*; D011817:Rabbits; D013379:Substrate Specificity
D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D007213:Indomethacin