L-Glutamine → O-acetyl-ADP-ribose

関与物質

反応手順

HeLa Cells were grown at 37° C. in Dulbecco's Modified Eagles Medium containing D-glucose 1 g/L, L-glutamine 1 g/L, pyridoxine hydrochloride 1 g/L, sodium pyruvate 110 mg/L, 10% fetal bovine serum (FBS), penicillin at 1000 units/mL, and streptomycin at 1 mg/mL (Gibco BRL) (growth medium). When cells had reached 80-90% confluency, they were rinsed once with PBS, lysed in 100-200 μL of ice-cold, 20 mM Tris, pH 7.2, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 100 μM phenylmethylsulfonyl fluoride (PMSF), 20 μg/mL leupeptin, and 20 μg/mL of aprotinin. Cells were scraped into eppendorf tubes, sonicated for 10s, and centrifuged at 16,000×g for 10 min to remove cell debris. Protein concentrations of supernatants were determined by the method of Bradford. Lysates (40 μL) of the cells or buffer controls were then incubated with radiolabeled 1-O-acetyl-ADP ribose (about 0.5 nmol) (total volume of reaction was 100 μL) for the indicated times prior to analysis by reverse phase HPLC. The HPLC analysis was carried out as described previously.

情報源と証拠