Comparison of the catalytic properties of phospholipase A2 from pancreas and venom using a continuous fluorescence displacement assay.
Phospholipases A2 from pig pancreas and the venoms from bee, Naja naja and Crotalus atrox have been studied by using a new continuous fluorescence displacement assay that utilizes normal phospholipid substrates [Wilton (1990) Biochem. J. 266, 435-439]. With limiting amounts of substrate, the assay demonstrated stoichiometric conversion into products with both pancreatic and venom enzymes, and thus would allow phospholipid determination at concentrations down to about 0.1 microM. The substrate specificity of the enzyme was determined for the four enzymes in terms of both phospholipid head group and fatty acid selectivity. None of the enzymes demonstrated a preference for arachidonic acid-containing phospholipid under the conditions of this assay. No lag was observed with any enzyme with either phosphatidylcholine or phosphatidylglycerol as substrate. With dipalmitoyl-phosphatidylcholine as substrate, the assay clearly highlighted the different membrane-penetrating properties of the pancreatic and Naja naja enzymes and demonstrated maximal activity for the pancreatic enzyme in the region of the phase-transition temperature of this substrate, at about 35 degrees C.
Kinkaid
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A
Department of Biochemistry, University of Southampton, U.K.
Wilton
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DC
D000818:Animals; D001514:Bee Venoms / Q000737:chemistry*; D002384:Catalysis; D003435:Crotalid Venoms / Q000737:chemistry*; D004546:Elapid Venoms / Q000737:chemistry*; D005227:Fatty Acids / Q000032:analysis; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000032:analysis / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature
D001514:Bee Venoms; D003435:Crotalid Venoms; D004546:Elapid Venoms; D005227:Fatty Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2