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. 2017 Aug 16;58(10):1999–2007. doi: 10.1194/jlr.M077792

Fig. 5.

Fig. 5.

Hydrolysis of (R)- or (S)-synthetic or bacterial Lipid 654 by various PLA2 lipid preparations. A: P. gingivalis Lipid 654 was aliquoted (10 μg per vial) and sonicated for 30 s (3 watts) in 10 mM Tris (pH 7.4) with the indicated concentrations of CaCl2. For samples with no CaCl2 added, the medium was supplemented with 5 mM EDTA (pH 7.4). The samples were stirred for 4 days at room temperature and were acidified with acetic acid followed immediately by extraction with CHCl3 (3 × 1 ml extractions). The extracts were dried and analyzed for levels of Lipid 654 and Lipid 430 using ESI-MRM. PP PLA2, HBV PLA2, LPL, and PLA1, PLC, and PLD (100 U each) were used for these hydrolyses. Results are depicted as mean ± SD (n = 2). Significant differences were noted for PP PLA2 hydrolysis or HBV hydrolysis of P. gingivalis Lipid 654 (*) versus controls (by one factor ANOVA with Fisher LSD pairwise comparisons). For (B) through (E), either bacterial or synthetic Lipid 654 was aliquoted (5 μg per vial) and sonicated for 30 s (3 watts) in 10 mM Tris (pH 7.4) with 10 mM CaCl. The samples were incubated for 3 days at room temperature unless otherwise noted, and were extracted and analyzed as described above. B: Time course of P. gingivalis Lipid 654 hydrolysis by PP PLA2 (100 U per sample). C: Stereoselective hydrolysis of synthetic (R)-Lipid 654 over synthetic (S)-Lipid 654 by either PP PLA2 or HBV PLA2. Results are depicted as mean ± SD (n = 3). Significant differences were noted for PP PLA2 hydrolysis or HBV hydrolysis of (R)-synthetic Lipid 654 [(R)-syn654] (*) versus (S)-synthetic Lipid 654 [(S)-syn654] and controls (by one factor ANOVA with Fisher LSD pairwise comparisons). D: Time course of human Rs PLA2 hydrolysis of (R)-syn654. (S)-syn654 is shown for comparison. E: Time course of human RLp PLA2 hydrolysis of P. gingivalis Lipid 654, (R)-syn654, and (S)-syn654. Results are depicted as mean ± SD (n = 2). Significant differences were noted for RLp PLA2 hydrolysis of P. gingivalis Lipid 654 (Pg 654) or (R)-syn654 versus (S)-syn654 (*) or between Pg 654 versus (S)-syn654 (#) (by one factor ANOVA with Fisher LSD pairwise comparisons).