Phospholipase A activities of the Cfp21 mutants.

<p>A) phospholipase A activity of the Cfp21 mutants: Hydrolysis of ¹⁴C-DPPC. FA: fatty acid; DPPC: dipalmitoyl-phosphatidylcholine; LysoPC: palmitoyl-lyso-phosphatidylcholine. Pure LysoPC, DPPC and Palmitic acid (FA) were used as standards (lanes 1, 2, and 3). Pancreatic porcine phospholipase A₂ (ppPLA₂) was used as a positive standard (lane 4). Products resulting from a 24-h enzymatic reaction with wild-type Cfp21 and its mutants were loaded into lanes 5 to 11. Rates of hydrolysis were calculated as described in the experimental section. B) Specific activities of phospholipase A₁ and A₂ on fluorescent phospholipids. The PLA₁ from <i>Thermomyces lanuginosus</i> was used as a positive standard to determine the PLA₁ activities, and ppPLA₂ was used as a positive standard to determine the PLA₂ activities. The specific fluorescent substrates used to measure the PLA₁ and PLA₂ activities were BODIPY® dye-labeled PED-A1 and the red/green BODIPY® PC-A2, respectively, as described in the Material and Methods section. Specific activities were calculated from the velocity slope obtained for 10 minutes using 10 µg of enzymes. Results are means obtained in at least 3 independent experiments.