Effect of sulfhydryl-alkylation and of disulfite bond formation on activity. Measurement of LPCAT1 activity was performed as described in legend of Figure 2. The standard deviation of at least 3 different measurements is indicated as error bars. (A) Treatment with diamide (0.5 mM) and N-ethylmaleimide (0.5 mM) were performed for 30 min before assaying acyltransferase activity. In some reactions, DTT was added at a concentration of 20 mM after treatment with diamide or NEM, and mixtures were incubated for another 10 min. Concentrations of the reagents were reduced 20-fold by dilution into the acyltransferase reaction mixture to assay the activity of the treated enzyme. Before assaying their activity, control samples were incubated for the same period of time and under the same condition in absence of diamide, of NEM and of DTT. The activity rate value obtained in absence of any of these chemicals (buffer without DTT) was arbitrary set at 1 and all others values were calculated relative to it. (B) Activity rate of LPCAT1 (circle) and of mutant C211T (triangle) were measured after treatment by different concentrations of NEM, as described above. For each protein, the value obtained in absence of treatment was set at 1 and values obtained after treatment were calculated relative to it. The activity rate ratio of the mutant form C211T, C(216,314,443,501,514)A obtained without and after treatment with 1 mM NEM is also reported (cross symbol).
Soupene and Kuypers BMC Biochemistry 2012 13:8 doi:10.1186/1471-2091-13-8