Reaktion #1005988

Reaktionsgleichung

11-deoxycorticosterone

DMSO

→

corticosterone

Lösungsmittel

ethanol

ethanol

Reaktionsbedingungen

Aufarbeitung

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

Vorschrift

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).

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