2 x 10^6 cells were transfected with 7.5 µg of the appropriate plasmid using 16 µl of jetPEI transfection reagent. In the experiments where HEK-293T cells were co-transfected with up to three different plasmids, the same number of seeded cells were transfected with 2.5 µg of each plasmid to reach a total of 7.5 µg plasmid DNA.

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (beta-alanyl-histidine) and homocarnosine (gamma-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic beta-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified approximately 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with beta-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed beta-alanyl-lysine, beta-alanyl-ornithine, gamma-aminobutyryl-lysine, and gamma-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on beta-alanyl-arginine and gamma-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from beta-alanine or gamma-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like alpha-alanyl-lysine and alpha-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was approximately 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (beta-alanyl-lysine, beta-alanyl-ornithine, gamma-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.