a. COS-1 cells transfected with either 1μg total DNA of wild-type ENPEP or ENPEP constructs with private coding sequence variants were grown in six-well plates. Following transfection, cells were incubated at room temperature for 4 hours before returning them to 37°C. After the indicated time intervals, the amount of cell surface activity of aminopeptidase A (APA) was assayed using α-glutamic acid ρ-nitroanilide as substrate. These assays showed that A676T had a gain of cell surface activity by 27% compared to wild-type, whereas E686K, R925G Q435E and E687D had decreased activity, with reductions of 71%, 61%, 30% and 23% respectively. E686K activity was comparable to that of untransfected cells. The D622N and K923I alleles were not distinguishable from wild-type. The results are the means +/-standard error of the mean (S.E.M) of triplicate measurements after independent transfections on the same day. IU: mmol hydrolysis of α-glutamic acid ρ-nitroanilide /min.
b. COS-1 cells transfected with either 1μg total DNA of wild-type or single nucleotide polymorphism (SNP) ENPEP constructs were grown in six-well plates. Following transfection, the cells were incubated at room temperature for 4 hours before returning them to 37°C. After the indicated time intervals, the amount of cell surface activity of aminopeptidase A (APA) was assayed using α-glutamic acid ρ-nitroanilide as substrate. The R159S plasmid had increased activity compared to wild-type (33%), whereas, W413X, E172Q and V218A had decreased cell surface activity (69%, 56% and 14% lower compared to wild-type respectively). Activity of the I32V, E172Q, Q213R, Y544F and S861R APA variants were not distinguishable from wild-type. The results are the means +/-standard error of the mean (S.E.M) of triplicate measurements after independent transfections on the same day. IU: mmol hydrolysis of α-glutamic acid ρ-nitroanilide /min.