XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
115.
IMMUNODETECTION OF 11SS-HYDROXYSTEROID DEHYDROGENASE
DURING PURIFICATION OF a NEW HUMAN MEMBraNE-BOUND
CarBONYL REDUCING ENZYME
Miloslava Netopilová, Libuše Černá, Lucie Škarydová and Vladimír Wsol
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University,
Hradec Králové , Czech Republic
The 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) is a membrane-bound microsomal
protein that mainly catalyzes the conversion of inactive glucocorticoid cortisone
to active cortisol. However, this enzyme has a broad substrate specificity and can also
metabolize various xenobiotics with carbonyl groups, for example the cytostatic drug
oracin. Oracin is reduced by microsomal enzymes to two enantiomers of 11-dihydrooracin
(DHO). Stereospecificity of the conversion depends on type of tissues and enzymes.
While human liver microsomes reduce oracin to (-)-DHO and (+)-DHO in a ratio of 60:40,
purified human liver 11ßHSD1 change oracin more specifically to (-)-DHO (76%). Because
11ßHSD1 is only known microsomal enzyme reducing oracin, it is supposed that the liver
microsomes contain at least one other oracin carbonyl reducing enzyme with stereospecific
production of (+)-DHO. The presence of 11ßHSD1 was determinated by immunodetection
using Western blotting. The blot was incubated with a primary polyclonal rabbit antibody
against human 11ßHSD1(1:1000, Abcam) and then with a secondary polyclonal swine
antibody anti-rabbit IgG coupled to horseradish peroxidase (1:1000, Dako). About 30
fractions were prepared using separation of human microsomes on Q-Sepharose. The
highest reducing activity was found in the flow through fractions Q2-Q4 and in the fractions
Q10-Q14; 11ßHSD1 was detected only in fraction Q11-Q14. The selected fraction
Q12 was separated on the Phenyl-Sepharose column and the highest activity was in the
fractions P10-P12. In these fractions, 11ßHSD1 was not found.
Acknowledgement: This project was supported by the Grant Agency of Charles University,
Grant No. 45508/C/2008.
240 XXII. Biochemistry Congress, Martin