The purification and partial characterization of phospholipase A2, a secretory protein of rabbit parotid gland.
Phospholipase A2 (EC 3.1.1.4), a soluble enzyme present in secretion granule lysates and in the discharged secretion of rabbit parotid gland, has been purified by gel filtration. The enzyme preparations obtained from both lysates and secretion have been found to have identical amino acid compositions, amino terminal residues (Asx), isoelectric points (10.2) and electrophoretic behavior in polyacrylamide gels. The reduced and alkylated protein yields a single band upon electrophoresis in the presence of sodium dodecyl sulfate; the mobility corresponds to an apparent molecular weight of 16000. The enzyme is capable of action on phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol. Activity has been examined using as substrate sonicated vesicles consisting of PC and PE. Rates of hydrolysis have been determined by densitometry of lysophosphatides resolved and charred on thin-layer chromatograms. This approach has been used to follow enzyme purification, to indicate the preferential hydrolysis (approx. 2-fold) of PE vs. PC and to demonstrate that enzyme purified from granule lysates and discharged secretion shows the same heat stability and activity profile as a function of pH. A highly specific Ca2+ requirement for activity also has been identified for substances organized as phospholipid bilayers; the apparent inactivity of this enzyme within a Ca2+-containing storage organelle, the secretion granule, presents an interesting problem for future investigation.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D010306:Parotid Gland / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D054467:Phospholipases A2; D011817:Rabbits
D000596:Amino Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium