Figure 4
From: Phosphatidylcholine formation by LPCAT1 is regulated by Ca2+ and the redox status of the cell
Cys-211 is not essential to the acyltransferase reaction. A collection of mutant forms in which C211 was changed to E, W, P, Y, G, Q, V, L, F and T was obtained by random mutagenesis of the Cys-211 codon. In addition, site-directed mutagenesis was performed to change C211 to S and R. Clones were tested for production of the mutant protein [panel A, top gel] and for their lysoPC-acyltransferase activity [panel A, bottom gel]. (A) Top gel: Proteins were separated on a 12% gel SDS-PAGE and stained with coomassie blue. Molecular mass standard (Precision Plus Protein Standard, Bio-Rad) is shown on the right (last lane). Position of the LPCAT1 protein (lane C) and of the mutant forms (S, R, E, W, P, Y, G, Q, V, L, F, T) is indicated with a black arrow on the left. Note the absence of LPCAT1 protein in the control sample prepared from cells containing the empty expression vector (lane vector). Production and position of the proteins were confirmed by Western blot detection of the hexa-histidine tags with an anti-histidine antibody (India-His, Pierce). (A) Bottom gel: Detection of the lysoPC-acyltransferase activity was performed with 5 μM [14C]-C18:1-CoA in presence of 20 μM LPC at 37°C with a 20 min incubation and with 10 μg of proteins, which represent 10 time more enzyme than the amount used to calculate activity rate (1 μg). Products were separated on silica plates and detected by phosphoimaging. Pure [14C]-PC was used as a migration standard, last lane on the right and position of [14C]-PC is indicated on the left with an arrow. Note the absence of the product [14C]-PC in the vector lane (left). (B) Activity rate measurements were performed as described in legend of Figure 2. LPCAT1, Rates of C211R mutant (see text) and of the C211 mutant forms able to form [14C]-PC, as shown on panel A, were determined. Values obtained for the mutant forms are reported relative to the activity rate obtained with the LPCAT1 enzyme (dubbed C211). Amino acid sequence of motif III is shown. The standard deviation of at least 3 different measurements is indicated as error bars.