SUBCELLULAR LOCALIZATION OF RAT GASTRIC PHOSPHOLIPASE A2

Abstract In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A 2 . After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A 2 was essentially enriched in the 105000 × g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A 2 activity (i.e., optimum of pH, calcium dependence, apparent K m and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A 2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A 2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A 2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A 2 , whereas acyl CoA-lysophosphatidylcholine; acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.