Quantitative Measurement of SAHH Activity

The experiments were performed in a 96-well scale using the Human Homocysteine (Hcy) ELISA Kit (Mybiosource, San Diego, CA, USA; MBS260128) that allows quantitative measurement of homocysteine concentration in cell extracts, according to the manufacturer's instructions. The concentration of homocysteine in cell extracts was used as a readout for in vivo SAHH activity,^{8 (link)} as SAHH hydrolyzes SAH to homocysteine and adenosine. Briefly, ARK2 cells were washed with cold phosphate-buffered saline and lysed on plate in 200 μl of lysis buffer (40 mm hexadecyltrimethylammonium bromide, 75 mm Tris-HCl, pH 8.0, 1m NaCl, 15 mm EDTA). The lysate was cleared of insoluble materials by centrifugation at 15 000 g at 4 °C for 15 min. Immediately following the centrifugation, 100 μl of the supernatant was collected and used for SAHH activity measurement. The absorbance of the samples was determined using a FilterMax F3&F5 Multi-Mode Microplate Reader (Molecular Devices).