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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
25
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pubmed:dateCreated |
2002-6-17
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pubmed:abstractText |
Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/17-Hydroxysteroid Dehydrogenases,
http://linkedlifedata.com/resource/pubmed/chemical/20-alpha-Dihydroprogesterone,
http://linkedlifedata.com/resource/pubmed/chemical/Androstenols,
http://linkedlifedata.com/resource/pubmed/chemical/Cross-Linking Reagents,
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Complementary,
http://linkedlifedata.com/resource/pubmed/chemical/Detergents,
http://linkedlifedata.com/resource/pubmed/chemical/Dihydrotestosterone,
http://linkedlifedata.com/resource/pubmed/chemical/Estradiol,
http://linkedlifedata.com/resource/pubmed/chemical/Estradiol Dehydrogenases,
http://linkedlifedata.com/resource/pubmed/chemical/Glycerol,
http://linkedlifedata.com/resource/pubmed/chemical/Histidine,
http://linkedlifedata.com/resource/pubmed/chemical/Liposomes,
http://linkedlifedata.com/resource/pubmed/chemical/NADP,
http://linkedlifedata.com/resource/pubmed/chemical/Oxygen,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Testosterone
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pubmed:status |
MEDLINE
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pubmed:month |
Jun
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pubmed:issn |
0021-9258
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
21
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pubmed:volume |
277
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
22123-30
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pubmed:dateRevised |
2009-11-19
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pubmed:meshHeading |
pubmed-meshheading:11940569-17-Hydroxysteroid Dehydrogenases,
pubmed-meshheading:11940569-20-alpha-Dihydroprogesterone,
pubmed-meshheading:11940569-Androstenols,
pubmed-meshheading:11940569-Animals,
pubmed-meshheading:11940569-Catalysis,
pubmed-meshheading:11940569-Cell Line,
pubmed-meshheading:11940569-Cell Membrane,
pubmed-meshheading:11940569-Centrifugation, Density Gradient,
pubmed-meshheading:11940569-Cross-Linking Reagents,
pubmed-meshheading:11940569-DNA, Complementary,
pubmed-meshheading:11940569-Detergents,
pubmed-meshheading:11940569-Dihydrotestosterone,
pubmed-meshheading:11940569-Dimerization,
pubmed-meshheading:11940569-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:11940569-Estradiol,
pubmed-meshheading:11940569-Estradiol Dehydrogenases,
pubmed-meshheading:11940569-Glycerol,
pubmed-meshheading:11940569-Histidine,
pubmed-meshheading:11940569-Humans,
pubmed-meshheading:11940569-Insects,
pubmed-meshheading:11940569-Kinetics,
pubmed-meshheading:11940569-Liposomes,
pubmed-meshheading:11940569-NADP,
pubmed-meshheading:11940569-Oxygen,
pubmed-meshheading:11940569-Protein Binding,
pubmed-meshheading:11940569-Protein Structure, Tertiary,
pubmed-meshheading:11940569-Recombinant Proteins,
pubmed-meshheading:11940569-Substrate Specificity,
pubmed-meshheading:11940569-Testosterone,
pubmed-meshheading:11940569-Time Factors
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pubmed:year |
2002
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pubmed:articleTitle |
Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2.
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pubmed:affiliation |
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUQ) and Laval University, Québec, Québec G1V 4G2, Canada.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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