With regard to the repair of oxidative damage to macromolecules, repair systems for DNA have been extensively studied,1 but recently there has been considerable interest directed toward repair of protein damage due to oxidation. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value Rabbit polyclonal to PLOD3 for a variety of research studies. Introduction Cells protect against oxidative damage by 2 general mechanisms, that is, both by destroying the reactive oxygen species (ROS) before damage can occur and by repairing the damage to the macromolecules after it occurs. Enzymes such as superoxide dismutase, catalase, and glutathione peroxidase can eliminate the ROS, and their role in protecting cells against oxidative damage is well established. With regard to the repair of oxidative damage to macromolecules, repair systems for DNA have been extensively studied,1 but recently there has been considerable interest directed toward repair of protein damage due to oxidation. One of the systems that has been extensively studied is the repair of methionine (Met) oxidation in proteins by the methionine sulfoxide reductase (Msr) system.2 Met is one of the most easily oxidized amino acids by ROS, being converted to methionine sulfoxide (Met(o)) as seen in effect of overexpression has been reported using cardiac myocytes. In that study, 13 cardiac myocytes were subjected to hypoxia and reoxygenation that caused cell death BMY 7378 due to oxidative damage. When these cells were transfected with adenovirus made up of the gene, significant protection of the cells from death was observed. What has drawn considerable attention was the finding that when MsrA was overexpressed in thioredoxin (Trx) and thioredoxin reductase (TrxB) were obtained from Dr. Todd Lowther, Wake Forest University School of Medicine. The recombinant proteins and bovine MsrA were overexpressed and purified from ribosomal protein3 or the reduction of free Met(o) using nitroprusside as a colorimetric reagent.23 The former assay is cumbersome, and the colorimetric assay is not BMY 7378 very sensitive. However, once it was apparent that this enzyme had a broad substrate profile and could reduce any compound made up of a methyl sulfoxide group, other assays were developed. A sensitive radioactive method was developed using for DMSO under the reaction conditions used in this study is usually 500?M, with maximum reaction velocity maintained at 5?mM and above (data not shown), the DMSO concentration would not be rate-limiting at compound concentrations at or above 10?6 M. illustrates the results (mean of 5 replicate experiments) using DMSO as substrate and 4 g of bovine MsrA. As shown in factors for the SeCm and NEM experiments are 0.95 (SD 0.003) and 0.92 (SD 0.007), respectively. These BMY 7378 values were determined at the 20-min time point, although there was little variation over the course of the experiment. These factors indicate that this assay is usually reproducible. In addition to the absorbance assay described earlier, there is also a fluorescence assay for NADPH. The fluorescence assay has been successfully used in an HTS format to screen for inhibitors of the redox cascade.28 Because NADPH is naturally fluorescent, emitting at 450?nm, while NADP is not, it would be relatively easy to switch to this type of assay. At present, we do not anticipate problems with the absorbance assay that cannot be controlled for, but if that should occur, we have also optimized conditions for a fluorescence-based NADPH assay (see Materials and Methods). shows the results of experiments using fluorescence to assay for the change in NADPH concentration dependent on MsrA, as well as the stimulation of the reaction by SeCm and the inhibition by NEM. As can be seen, there is a significant stimulation by SeCm and inhibition by NEM, which closely parallels the.