- Synthesis of [3α-3H]-Dehydroepiandrosterone and [3α-3H]-pregnenolone
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[3α-3h]-dehydroepiandrosterone ([3α-3h]-3β-hydroxy-5-androsten-17- one) and [3α-3h]-pregnenolone ([3α-3h]-3β-hydroxy-5-pregnen-20-one) were prepared by selective reduction of 3-keto-5-ene intermediates with tritiated sodium borohydride. These were used as substrates to set up a tritium release assay for 3β-hydroxysteroid oxido-reductase and 5→4-ene isomerase (3β- HSD) which is a key enzyme in steroidogenesis.
- Tait
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- Δ5-3β-hydroxysteroid dehydrogenase (3βHSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme
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During the biosynthesis of cardiac glycosides, Δ5-3β- hydroxysteroid dehydrogenase (3βHSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3β-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3βHSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3βHSD was isolated by RT-PCR from the same source. A Sph I/Kpn I 3βHSD cDNA was cloned into the pQE30 vector and then transferred into E. coli strain M15[pREP4]. 3βHSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3βHSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). PQ3βHSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3β-hydroxypregnanes but not cholesterol were 3β-oxidised by pQ3βHSD when NAD was used as the co-substrate. Testosterone (4-androsten-17β-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3βHSD has also 17β-dehydrogenase activity. pQ3βHSD was able to reduce 3-keto steroids to their corresponding 3β-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3βHSD genes were isolated and sequenced from another 6 species of the genus Digitalis. Gene structure and the deduced 3βHSD proteins share a high degree of similarity. Georg Thieme Verlag KG Stuttgart.
- Herl, Vanessa,Frankenstein, Joerdis,Meitinger, Nadine,Mueller-Uri, Frieder,Kreis, Wolfgang
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