Assaying phospholipase A2 activity.
Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2alpha(cPLAalpha) and for secreted PLA2s (sPLA2) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA2alpha involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA2alpha activity in cell lysates from other PLA2s. sPLA2 activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.
Leslie
Christina C
CC
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA.
Gelb
Michael H
MH
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054522:Group VI Phospholipases A2; D006801:Humans; D007553:Isotope Labeling; D010741:Phospholipases A / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D002457:Cell Extracts; D005227:Fatty Acids; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D054467:Phospholipases A2