Ac-SDKP hydrolysis by recombinant mutant (S333W) ACE.

<p><b>A</b>. ACE activity of the membrane-bound form of ACE of CHO cells (lysates) stably expressing WT and mutant ACE was determined with substrates Hip-His-Leu and Z-Phe-His –Leu (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088001#pone-0088001-g008" target="_blank">Fig. 8</a>) and with natural substrate Ac-SDKP (see Experimental Procedures). ACE activity (mU/ml of the lysate) was expressed as % of WT. <b>B–C</b>. The lysates of these cells (normalized by loading of 10 µg protein per lane) were subjected to SDS-PAGE (4-15% gradient gel) in reducing conditions for Western blotting (<b>B</b>) or protein quantification (<b>C</b>). <b>B</b>. Western blotting was performed with rat mAb 4G6 that recognizes the denatured epitope on the C-domain of mouse and human ACE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088001#pone.0088001-Balyasnikova4" target="_blank">[50]</a>. Proteins transferred on PVDF-Plus membrane were revealed with 1/5 dilution of culture medium from hybridoma cells producing mAb 4G6. Molecular weight markers are shown by arrows on the left of panel B, which is a representative experiment. <b>C</b>. The relative amount of WT and mutant ACE revealed by Western blotting with mAb 4G6 (<b>B</b>) was quantified by the image analysis (densitometry) using ImageJ software (NIH). Data are expressed as mean ± SD of 2 independent measurements. <b>D</b>. ACE activity in the lysates of CHO cells stably transfected with WT and mutant ACE towards natural substrate Ac-SDKP 0.75 mM (A) was normalized to the amount of ACE protein in the lysate, determined by densitometry (C) after Western blotting (B).