Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta.
Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.
Lin
S X
SX
Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de l'Université Laval Research Center, Quebec, Canada.
Yang
F
F
Jin
J Z
JZ
Breton
R
R
Zhu
D W
DW
Luu-The
V
V
Labrie
F
F
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015067:17-Hydroxysteroid Dehydrogenases