Isolation of purified brush-border membranes from rat jejunum containing a Ca2+-independent phospholipase A2 activity.
A novel phospholipase activity was recognized in intact, rat jejunal brush-border membranes and its effect on membrane lipid composition was evaluated following various incubation protocols. Brush-border membranes were isolated from mucosal scrapings by a combination of existing techniques. A brush-border plus nuclei fraction was first prepared by homogenization and low-speed centrifugation in isotonic mannitol, in the presence of 5 mM EDTA. Brush-border membrane vesicles were isolated from this fraction by homogenization, followed by precipitation of the remaining undesired membranes with 10 mM CaCl2. Membranes were judged to be highly purified by marker enzyme content, protein profile, and electron microscopy. In total lipid extracts, prepared immediately following membrane isolation, the ethanolamine phosphatides were found to be the major phospholipid class, accounting for nearly 45% of the total lipid phosphorus. Storage of the intact membranes, at either room temperature or at -20 degrees C, but not at -70 degrees C, resulted in a gradual and progressive hydrolysis of phosphatidylethanolamine to lysophosphatidylethanolamine. Over 60% of the total ethanolamine phospholipid was converted to the lyso form during a 2 week storage period. Incubation of the intact membranes at 37 degrees C produced a similar effect in one hour. Only small amounts of other glycerophospholipids were degraded under these conditions. Hydrolysis was specific for the sn-2 position as more than 80% of the fatty acids in the lysophosphatidylethanolamine were found to be saturated. Substitution of MgCl2 for CaCl2 in the precipitation step did not block the hydrolysis. It was concluded that rat brush-border membranes contain a Ca2+-independent phospholipase A2 with a high substrate preference for phosphatidylethanolamine. The physiological significance of this enzyme is not known.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D004492:Edetic Acid / Q000494:pharmacology; D007583:Jejunum / Q000648:ultrastructure*; D008066:Lipolysis; D008246:Lysophospholipids; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism; D008854:Microscopy, Electron; D008871:Microvilli / Q000201:enzymology* / Q000648:ultrastructure; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D008246:Lysophospholipids; D008563:Membrane Lipids; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C008301:lysophosphatidylethanolamine; D004492:Edetic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium