Reaction #1005988

Reaction equation

11-deoxycorticosterone

DMSO

→

corticosterone

Solvents

ethanol

ethanol

Conditions

Workup

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

Procedure

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).

Source