Figure 1. Scpep1 has carboxypeptidase activity against ET-1.

A. Carboxypeptidase activity against ET-1 in homogenates of cultured AVSMC from WT, Scpep1 ^−/−, CathA^S190A and Scpep1 ^−/− /CathA^S190A mice as well as from the cells of Scpep1 ^−/− /CathA^S190A mice transiently transfected with pCMV-GFP and pCMV-Scpep1 plasmids. AVSMC after the passage 3 were transfected or not with Scpep1-RGS-His-Tag and pEGFP-C1 plasmids. Forty-eight hours after transfection confluent cells were harvested, homogenized and tested for carboxypeptidase activity using 50 µM ET-1 as a substrate. Data are expressed as means (±S.D.) of 3 independent experiments performed with different cell cultures. * p<0.05 and ** p<0.01 in two-tailed unpaired t-test. B. Carboxypeptidase activity against ET-1 in homogenates of cultured AVSMC from WT mice transfected with Scpep1 and CathA shRNA. AVSMC after the passage 3 were transfected with pRFP-C-RS vectors expressing shRNA for mouse CathA and Scpep1 or non-effective 29-mer scrambled shRNA as indicated. Seventy-two hours after transfection the cells were harvested, and those expressing plasmids enriched by cell-sorting, homogenized and tested for carboxypeptidase activity using 50 µM ET-1 as a substrate. Data are expressed as means (±S.D.) of 3 independent experiments performed with different cell cultures. ** Significantly different from non-transfected or scrambled shRNA-transfected cells; p<0.01 in two-tailed unpaired t-test. C. Carboxypeptidase activity against ET-1 of purified recombinant Scpep1. Scpep1 carrying a His6 tag at the C-terminus was expressed in stably transfected HT1080 cells and purified by affinity chromatography on Ni-NTA resin followed by anion-exchange chromatography as described [19]. Carboxypeptidase activity was assayed with 50 µM ET-1 as a substrate and 0.4–0.8 µg of the purified enzyme in the reaction mixture in 50 mM sodium phosphate/50 mM sodium acetate buffer at pH 5.5, 6.5 and 7.5. Bars show mean values (±S.D.) of 3 independent experiments. In separate experiments we confirmed that under the conditions used the amount of liberated amino acid was directly proportional both to the incubation time and to the amount of Scpep1 protein in the reaction mixture (inset graphs). The amino acid analysis of the reaction mixture determined that Scpep1 cleaves the C-terminal amino acid (Trp21) from ET-1 (not shown).