反応 #1005988

反応方程式

11-deoxycorticosterone

DMSO

→

corticosterone

溶媒

ethanol

ethanol

反応条件

後処理

1. 1
   workup.ADDITIONA suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml
2. 2
   その他was prepared on a freshly grown culture
3. 3
   その他The enzyme reaction
4. 4
   その他The test was quenched
5. 5
   抽出by extracting the sample with 500 μl of EtOAc
6. 6
   その他After centrifugation (10,000 g, 2 min), the EtOAc phase was removed
7. 7
   その他evaporated to dryness
8. 8
   その他The reaction of the substrate

実験手順

A suspension of fission yeast (S. pombe PE1) with a cell density of 3·107 cells/ml was prepared on a freshly grown culture using fresh EMMG (pH 7.4) as modified according to Ehmer et al. (Ehmer, P. B. et al., 1. Steroid. Biochem. Mol. Biol. 81, 173-179 (2002)). 492.5 μl of this cell suspension was admixed with 5 μl of inhibitor solution (50 μM of the compound to be tested in ethanol or DMSO) and incubated at 32° C. for 15 min. Controls were admixed with 5 μl of ethanol. The enzyme reaction was started by adding 2.5 μl of 11-deoxycorticosterone (20 μM, containing 1.25 nCi of [4-14C]11-deoxycorticosterone in Ethanol), followed by horizontal shaking at 32° C. for 6 h. The test was quenched by extracting the sample with 500 μl of EtOAc. After centrifugation (10,000 g, 2 min), the EtOAc phase was removed and evaporated to dryness. The residue was taken up in 10 μl of chloroform. The reaction of the substrate to form corticosterone was analyzed by HPTLC (see below).

出典