adenosylhomocysteine hydrolase activity in biolog

Supplementary information

A fluorescence-based assay for the measurement of Sadenosylhomocysteine hydrolase activity in biological samples

Roman Hudec1, Kozo Hamada1,2, and Katsuhiko Mikoshiba1,2,#

Supplementary methods

Crosslinking of purified SAHH and detection by silver staining Mass of 250 ng of purified HA-SAHH (in 70 mM potassium phosphate (pH 7.2), 100 mM NaCl, and 1 mM EDTA) was supplemented with EDTA (to a final concentration of 6 mM) and incubated in the presence of 0 - 500 μM of the bismaleimidohexane (BMH) crosslinker (Pierce) (10X stock solution in DMSO) for 30 min on ice. The reaction was performed in a total volume of 10 μl, stopped by the addition of 4X SDS-PAGE sample buffer and incubated for 15 min at 55°C. The samples were then subjected to SDS-PAGE (3% stacking gel and 6% separating gel) followed by protein detection using a silver staining reagent kit (Daiichi Pure Chemicals). The Precision Plus Protein Standards (Bio-Rad) were used as the molecular weight markers.

Crosslinking of endogenous and overexpressed SAHH in cell lysates The non-transfected and flag-SAHH-transfected HeLa cells were grown on 6-cm dishes, washed twice with PBS, harvested in 1 ml of ice-cold PBS on ice, and centrifuged at 14,000 × g and 4°C for 10 min. The cell pellet was re-suspended in lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, and 2 mM EDTA) and sonicated five times in 10 s cycles. The supernatant was collected by centrifugation at 3,800 × g and 4°C for 10 min. The concentrations of the proteins were determined through the Bradford method using the protein assay reagent (Bio-Rad). For the crosslinking, a total protein

concentration of 10 - 20 μg in lysis buffer was supplemented with EDTA (to a final concentration of 7 mM) and 10X concentrated BMH crosslinker in DMSO. The mixture was incubated for 30 min on ice. The reaction was stopped by the addition of 4X SDS-PAGE sample buffer and incubated for 15 min at 55°C. The samples were then subjected to SDS-PAGE and immunoblotting.

Immunocytochemistry The HeLa cells were grown in 4-well chamber slides (Nunc) and transfected with SAHH siRNA and mRFP, which was used as a marker for the SAHH-knockdown cells. The cells were fixed with 4% formaldehyde in PBS 48 h post-transfection and permeabilized with 0.1% Triton X-100 in PBS. The samples were blocked with 2% NGS (Vector Laboratories) in PBS for 1 h at RT and then incubated with anti-SAHH antibody for 1 h at RT. The slides were washed and then incubated for 1 h with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen). After washing, the samples were mounted with Vectashield medium containing DAPI (Vector Laboratories). The images were generated using a confocal microscope (Leica).

Application of SAHH inhibitor to the cell culture The treatment of HeLa cells with the SAHH inhibitor 3-deaazaadenosine (3-DZA, Sigma) was conducted as follows: The HeLa cells were grown to 80% confluence under the standard conditions described in “Materials and Methods”. The 3-DZA was added to the cells to attain final concentrations of 0 (control), 40 or 80 µM. The cells were incubated in the presence of the inhibitor for 8 h and subsequently used in the SAHH activity assay, as described in “Material and Methods”.

Figure S1. The crosslinking of the purified and cell-expressed SAHH proteins demonstrates the oligomeric state of SAHH. (A) 250 ng of purified HA-SAHH were crosslinked with BMH, separated by SDS-PAGE and silver stained. The molecular weight markers were run in the first line, which is marked as M. (B and C) Immunodetection of the SAHH profile in HeLa cell lysates upon crosslinking with BMH. (C) Lysates with total protein amounts of 10 μg (left) and 20 μg (right) were used for the crosslinking. The endogenous SAHH was detected using an anti-SAHH antibody. (D) Lysates with a total protein amount of 20 μg were used. The overexpressed flag-SAHH was detected using an antiflag antibody. The concentrations of the BMH crosslinker (in μM) and the protein molecular weight (in kDa) are indicated at the top and left of each figure, respectively. The positions of the SAHH monomer (I), dimer (II) and tetramer (IV) are indicated on the right side of each figure. The detected signal, which is marked with a single asterisk (*), corresponds to the putative SAHH hexamer. Two asterisks (**) mark the signal exhibited by the cell lysates only. Figure S2. Specificity of the SAHH detection in HeLa cells. (A) An anti-SAHH antibody was used for the detection of SAHH in HeLa cells transfected with control and three different SAHHspecific (No. 1, 2, and 3) siRNAs. Lysates with 15 μg total protein were used in each line. The β-actin levels were used as the loading control. (B) Representative confocal images of non-transfected and transfected HeLa cells are shown. The HeLa cells were transfected with mRFP and SAHH-specific (No. 1) siRNA. The expression of SAHH was detected using an anti-SAHH antibody (green), and mRFP was used to detect the transfected cells (red). The nuclear staining by DAPI is shown in blue. The images of both groups of cells were obtained under the same conditions. Scale bar, 5 m. Figure S3. Specific SAHH activity in HeLa cells treated with the SAHH inhibitor. The HeLa cells were treated with the indicated concentrations of the SAHH inhibitor 3-deazaadenosine (3-DZA) for 8 h. Lysates with 80 µg total protein were subjected to the SAHH activity assay. The specific activities of the endogenous SAHH were calculated as described in “Materials and Methods”. The average specific SAHH activity in the control (non-treated) cells was 0.12 ± 0.02 U/mgtotal prot. The presence of 40 µM and 80 µM 3-DZA caused an inhibition of the endogenous SAHH activity of nearly 40% (0.07 ± 0.01 U/mgtotal prot) and almost 53% (0.06 ± 0.01 U/mgtotal prot). *p= 0.0026 and **p= 0.0021. The inset shows the SAHH kinetics at the given conditions: control, “open circle”; 40 µM 3-DZA, “open square”; and 80 µM 3-DZA, “closed square”. The lines represent the fitting of the obtained data with Eq. 1.