Characterization of molecular and catalytic properties of intact and truncated human 17beta-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization of the wild-type enzyme in the endoplasmic reticulum.
Human 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17beta-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20alpha-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5beta-androstan-3alpha,17beta-diol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3beta,17beta-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17beta-hydroxysteroids.
Puranen
T J
TJ
Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, Finland.
Kurkela
R M
RM
Lakkakorpi
J T
JT
Poutanen
M H
MH
Itäranta
P V
PV
Melis
J P
JP
Ghosh
D
D
Vihko
R K
RK
Vihko
P T
PT
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D002384:Catalysis; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution / Q000502:physiology
D007527:Isoenzymes; D010446:Peptide Fragments; D015067:17-Hydroxysteroid Dehydrogenases