The purification and characterization of a phospholipase A in hamster heart cytosol for the hydrolysis of phosphatidylcholine.
Phospholipases A1 and A2 catalyze the hydrolysis of acyl groups of phospholipids at C-1 and C-2, respectively. These phospholipases are important in phospholipid catabolism and the remodeling of the acyl groups of phospholipids. Phospholipase A from hamster heart cytosol was purified by a combination of ion-exchange and gel filtration chromatography. The purity of the enzyme was assessed by nondenaturing polyacrylamide gel electrophoresis, two-dimension polyacrylamide gel electrophoresis, and immunological studies. The purified enzyme exhibited both phospholipase A1 and A2 activities toward phosphatidylcholine and had the ability to hydrolyze the acyl groups of phosphatidylethanolamine. However, the enzyme was not active toward lysophosphatidylcholine, diacylglycerol, or triacylglycerol. By Sepharose 6B chromatography, the molecular weight of the purified enzyme was estimated to be 140,000. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of identical Mr 14,000 subunits. At least six subunits in the native enzyme could be cross-linked by dimethyl suberimidate. Both phospholipase A1 and A2 activities showed similar pH profiles, exhibited no absolute requirements for divalent metallic cations, but displayed a high degree of specificity for the acyl groups of phosphatidylcholine at both C-1 and C-2. The Km of phospholipases A1 and A2 for 1-palmitoyl-2-arachidon-ylglycerophosphocholine was found to be identical (0.5 mM).
D000818:Animals; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008647:Mesocricetus; D009206:Myocardium / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D054466:Phospholipases A1; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1