Figure 2

Role of D³⁹²and E⁴⁰³in Ca²⁺-inhibition of LPCAT1 activity. Asp-392 and Glu-403 of the predicted EFh-1 motif of LPCAT1 were changed by site-directed mutagenesis to an alanine, and the activity of the three mutants enzymes, D³⁹² A, E⁴⁰³ A and D³⁹²A,E⁴⁰³A was determined. Activity measurements were performed with 5M [¹⁴ C]-C_18:1 -CoA in the presence of 20 μM LPC at 37°C with 1μg of proteins. Acylation rates were calculated between 0 and 8 min. The standard deviation of 3 different measurements is indicated as error bars. After separation by thin-layer chromatography, the amount of [¹⁴C]-PC formed during the reaction was quantified by phosphoimaging (see the Experimental section) and the activity rate values are calculated as PC formed/ μg of protein per min. (A) Measurement of the activity rates was performed with LPCAT1 and the 3 mutant enzymes. Values obtained for the mutant forms are reported relative to the activity rate value obtained with LPCAT1. (B) Activity of LPCAT1 and of the three mutant forms was determined in the absence and in the presence of 10 mM calcium chloride. Activity rates were calculated as described above. The ratio of the value obtained in presence of Ca²⁺ relative to the value obtained in its absence for each protein is reported as fold inhibition. Note that a ratio value of 1 indicates that the activity rate in presence and in absence of Ca²⁺ was identical.