Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins.
The effects of human recombinant lipocortin I (annexin I) and bovine lung calpactin I (annexin II) on porcine pancreatic phospholipase A2 (PLA2) activity in phosphatidylcholine (PC)/deoxycholate (DOC) mixtures were investigated. Annexin-associated decreases in PLA2 activity were observed under some conditions, for example, at high DOC/PC molar ratios; however, activation was observed under other conditions. NaCl, which lowers the non-critical micellar concentration (NCMC) of deoxycholate, caused significant decreases in control PLA2 activity in the absence of annexins, and greater decreases in PLA2 activity in annexin-containing samples, resulting in an apparent increase in inhibition. The PC/DOC substrate mixtures themselves appeared unstable. Despite a large excess of detergent, precipitates were, at times, observed upon incubation of some PC/DOC mixtures at 37 degrees C. Such behavior is of interest in view of the numerous reports of PLA2 inhibition by annexins and annexin-derived peptides in the PC/DOC system. The influence of the annexins on activity in this system is consistent with effects on the phase behavior of the PC/DOC mixture and/or competition with the enzyme for available Ca2+. These results caution against use of the PC/DOC system for analysis of potential PLA2 inhibitors unless the phase behavior of the system is more fully delineated.
Davidson
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
Dennis
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EA
D000818:Animals; D017302:Annexins; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D003840:Deoxycholic Acid; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000494:pharmacology; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000494:pharmacology; D013552:Swine
D017302:Annexins; D002135:Calcium-Binding Proteins; D002250:Carbon Radioisotopes; D008565:Membrane Proteins; D009829:Oleic Acids; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D003840:Deoxycholic Acid; D019301:Oleic Acid; D010741:Phospholipases A; D054467:Phospholipases A2