反应详情

Prepare substrate solution A containing 8 mM adenosine triphosphate disodium (ATP), 32 mM DL-methionine, 100 mM Tris hydrochloride buffer, 100 mM KCl (final concentration) and 20 mM MgCl2 (final concentration) and adjusted pH to 7.5. Two hundred and fifty μl of the prepared substrate solution A was mixed with 150 μl sterile distilled water, 100 μl of the S-adenosylmethionine synthetase prepared according to Example 7, incubated at 58° C. for 20 min. The reaction was stopped by adding 300 μl 10% trichloroacetic acid (TCA), and then centrifuged at 17,800 g for 15 min at 10° C. to collect the supernatant as S-adenosylmethionine. The quantity of S-adenosylmethionine was determined by high pressure liquid chromatography (HPLC). HPLC column: C18 reverse column (Waters, USA); mobile phase: acetonitrile:water (85:15); flow rate: 0.5 mL/min; detector: ELSD 500. Injection volume was 10 μl. Protein concentration was determined using Coomassie® Plus Protein Assay Reagent Kit (Pierce, USA) and SDS-PAGE. One unit of enzyme specific activity is defined as the amount of enzyme that produces S-adenosylmethionine from 1 μmole of adenosine triphosphate (ATP) and 1 μmole of DL-methionine per min under the above described conditions. Table 2 below shows the specific activity of wild-type S-adenosylmethionine synthetase.

1. concentrationconcentration) and 20 mM MgCl2 (final
2. concentrationconcentration) and
3. additionTwo hundred and fifty μl of the prepared substrate solution A was mixed with 150 μl
4. distillationsterile distilled water, 100 μl of the S-adenosylmethionine synthetase
5. customprepared
6. waitcentrifuged at 17,800 g for 15 min at 10° C.
7. customto collect the supernatant as S-adenosylmethionine
8. concentrationProtein concentration