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  • 1
    Electronic Resource
    Electronic Resource
    Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism? (1999)
    Springer
    Planta 209 (1999), S. 478-486 
    Details
    ISSN: 1432-2048
    Keywords: Key words:Δ5-3β-Hydroxysteroid dehydrogenase ; Δ5-Δ4-Ketosteroid isomerase ; Pregnenolone ; Proges-terone ; Short-chain dehydrogenase/reductases ; Digitalis (cardenolide biosynthesis)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Δ5-3β-Ηydroxysteroid dehydrogenase (Δ5-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ5-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ5-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004250050751
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  • 2
    Electronic Resource
    Electronic Resource
    Purification and characterization of lanatoside 15′-O-acetylesterase from Digitalis lanata Ehrh. (1998)
    Springer
    Planta 204 (1998), S. 383-389 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Acetylesterase ; Cardenolide ; Cell wall ; Digitalis ; Lanatoside 15′-O-acetylesterase ; Somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s−1 · (g protein)−1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s−1 · (g protein)−1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004250050270
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    Paper (German National Licenses)
    Fulltext
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