Specificity of PLAT1 for acyl-CoAs (A) and LPLs (B, C).

<p>(A) FLAG-PLAT1 was purified using the ANTI-FLAG M2 Affinity Gel and 3X FLAG peptide to remove the endogenous LPLs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102377#s2" target="_blank">Materials and Methods</a>. Each assay contained 25 µM acyl-CoA, 1 µM [1-¹⁴C]palmitoyl-LPC, and purified enzyme (10 µl of gel suspension) in 100 µl of 100 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.01% sodium cholate. The reaction was conducted at 30°C for 20 min and terminated by the addition of 500 µl of CHCl₃/CH₃OH (2∶1, v/v). The reaction mixture was loaded on the TLC plate and developed with CHCl₃/CH₃OH/H₂O (65/25/4, v/v/v). The radioactivity of the band corresponding to PC on the TLC plate was quantified by a FLA 5100 Bio-imaging analyzer. Data represent the mean ± SD (n = 3). (B) Each assay contained 1 µM of LPLs, 1 µM [1-¹⁴C]palmitoyl-CoA, and the purified enzyme (10 µl of gel suspension) in 100 µl of 100 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 0.01% sodium cholate. Mock represents the experiment using the enzyme prepared from the lysate of yeast cells harboring the empty vector. The assay was conducted at 30°C for 20 min and terminated by the addition of 500 µl of CHCl₃/CH₃OH (2∶1, v/v). The reaction mixture was loaded on the TLC plate, developed with CHCl₃/CH₃OH/H₂O (65/25/4, v/v/v), and analyzed with a FLA 5100 Bio-imaging analyzer. The typical TLC is presented here. (C) The enzyme reaction was performed by the same procedure as shown in (B). The reaction products were applied on TLC plates and the radioactivity of the band corresponding to each PL was quantified by a FLA 5100 Bio-imaging analyzer (n = 3). Data represent the mean ± SD.