Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase |
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Authors: | Laura A. Wittenauer Kohji Shirai Richard L. Jackson J.David Johnson |
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Affiliation: | Division of Lipoprotein Research Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine Cincinnati, Ohio 45267 USA |
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Abstract: | The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the -1 and -2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme. |
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Keywords: | 1-acyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine LpL bovine milk lipoprotein lipase apolipoprotein C-II apoC-II CMC critical micellar concentration |
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