Figure 10.

Characterization of YKL094W, a homolog of AhMGAT. YKL094W was purified by Ni²⁺-NTA column chromatography, and the enzyme assays were performed with dialyzed protein. A, Protein-dependent hydrolysis of [¹⁴C]MAG with increasing amounts of protein for 10 min. Lane 0, Enzyme was added after stopping the reaction. FFA, Free fatty acid. B, Time-dependent MAG hydrolysis assay was performed at different time intervals at 30°C. The reaction was stopped by the addition of chloroform:methanol:2% orthophosphoric acid (1:2:1, v/v). Lipids were extracted and separated on a silica-TLC plate using petroleum ether:diethyl ether:acetic acid (70:30:1, v/v) as the solvent system. C, The time-dependent LPC hydrolase assay was conducted using [¹⁴C]LPC with 2 µg of purified protein. Lane 0, Enzyme was added after stopping the reaction; lane T₀, zero time point (reaction was stopped immediately after adding the enzyme). D, The LPC hydrolase assay was performed for 10 min at 30°C in the presence of increasing amounts of purified YKL094W. The reaction was stopped, and lipids were resolved on a TLC plate using chloroform:methanol:28% ammonia (65:25:5, v/v) as the solvent system. Values are means ± sd of at least three independent experiments.