Detection of sulfhydryl-modification of cysteine mutant forms. Purified proteins (panel A) and membrane fractions obtained from cells producing LPCAT1 and several Cysteine mutant forms (panel B) were treated with maleimidylpropionyl-biocytin (MPB) as described in the Experimental section. (A) Proteins were purified and 2 μg aliquots were labeled with 100 μM of MPB protected from light. Reactions were stopped by quenching the unreacted MPB reagent with 25 mM DTT. Labeled and unlabeled proteins were separated on a 12% gel SDS-PAGE, and visualized by coomassie blue staining (bottom gel). Proteins were transferred onto PVDF membranes and blotted with HRP-conjugated streptavidin, which interacts with the biotin group of MPB. Molecular mass standard is shown on the right. (B) An equivalent of 25 μg of microsomal proteins as shown in Figure 4A and Figure 6A were labeled with 100 μM MPB. After quenching with DTT, hexa-histidine tagged LPCAT1 and mutant forms were affinity-purified. Proteins were separated on a 12% gel SDS-PAGE, transferred onto PVDF membranes and blotted with HRP-conjugated streptavidin (top membrane) or with a HRP-conjugated anti-histidine antibody (bottom). The mutant form lacking all 12 cysteines (Cys12-) was detected (last lane, bottom) but was not labeled (last lane, top). The mutant form C211T, C(216,314,443,501,514)A, whose activity was not inhibited by treatment with NEM , lane NEMr, was labeled by MPB. Results obtained with clone FK605, FK607, FK606, FK609, FK613 and FK614 (see Additional file 2: Table S1) are shown in lane Cys7-, Cys8-, Cys9-, Cys10-, Cys11- and Cys12-, respectively. Molecular mass standard is indicated on the right.
Soupene and Kuypers BMC Biochemistry 2012 13:8 doi:10.1186/1471-2091-13-8