O-Methylierung von Adrenalin, 3.4-Dihydroxybenzoesäure und 6.7-Dihydroxycumarin in Sproßpilzen /O-Methylation of Epinephrine, 3,4-Dihydroxybenzoic Acid and 6,7-Dihydroxycoumarin in Yeasts

1976 ◽  
Vol 31 (9-10) ◽  
pp. 509-513 ◽  
Author(s):  
D Müller-Enoch ◽  
H Thomas ◽  
W Streng ◽  
W Ildfeuer ◽  
O Haferkamp
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Isotope Dilution ◽  
Dihydroxybenzoic Acid ◽  
Dilution Technique ◽  
Isotope Dilution Technique ◽  
Methyl Derivatives ◽  
Layer Chromatography ◽  
Methoxybenzoic Acid

Abstract In the yeasts C. albicans, C. tropicalis, and C. stellatoidea but not in C. krusei, R. rubra, and S. cerevisiae enzyme activity was found by which - as by the catechol-O-methyltransferase (EC 2.1.1.6) found in the liver - the O-methylation of epinephrine to metanephrine and paranephrine, of 3,4-dihydroxybenzoic acid to 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid, and of 6,7-dihydroxycoumarin to 7-hydroxy-6-methoxycoumarin and 6-hydroxy-7-methoxy-coumarin is catalysed. When the substrates 3,4-dihydroxybenzoic acid, or 6,7-dihydroxycoumarin or epinephrine were incubated in the presence of S-adenosyl-ʟ-[methyl-14C] methionine and S-adenosylmethionine hydrogensulfate with a 100 000 X g supernatant of C. albicans, C. tropicalis or C. stellatoidea the cor­responding O-methylethers were detected in the extracts of the incubation medium by thin-layer chromatography. Final identification of the isomeric radioactive O-methylethers obtained from 3,4-dihydroxy­ benzoic acid and 6,7-dihydroxycoumarin was performed after thin-layer chromatographic separation by the reversed isotope dilution technique. The radioactive m-and p-O-methyl derivatives from epinephrine were separated by thin-layer chromatography and then cleaved with periodate to the corresponding aldehydes which were also identified mainly by the reversed isotope dilution technique.

Metabolisierung von 3.4-Dimethoxybenzaldehyd und 3.4-Dimeth- oxybenzoesäure in der isoliert perfundierten Rattenleber / Metabolism of 3,4-Dimethoxybenzaldehyde and 3,4-Dimethoxybenzoic Acid in Perfused Rat Liver

1974 ◽  
Vol 29 (9-10) ◽  
pp. 602-607 ◽  
Author(s):  
D. Müller-Enoch ◽  
H. Thomas ◽  
P. Holzmann ◽  
K. Haider ◽  
H. Harms
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Methoxy Group ◽  
Specific Activity ◽  
Aldehyde Group ◽  
Perfused Rat Liver ◽  
Dilution Technique ◽  
Rat Livers ◽  
Layer Chromatography ◽  
Methoxybenzoic Acid

Abstract Isolated rat livers were perfused with 3,4-dimethoxybenzaldehyde and 3,4-dimethoxybenzoic acid, which were 14C-labelled in the m eta-and/or para-configurated methoxy group. Because the aldehyde group was completely oxydized to the carboxylic acid, the perfusions with 3,4-dimethoxybenzalde­ hyde and those with 3,4-dimethoxybenzoic acid exhibited principally the same results. After per­ fusion with a 1:1 mixture of (4 -O -[14C]methyl)-and (3 -O -[14C]methyl)-3,4-dimethoxybenzalde­ hyde, each of which had the same specific activity, thin layer chromatography of an extract of the perfusate revealed both radioactive 4-hydroxy-3-methoxybenzoic acid and radioactive 3-hydroxy-4-methoxybenzoic acid as demethylation products. The distribution of radioactivity between these two metabolites was determined as 20:1. Thus, demethylation of the 3,4-dimethoxyphenyl com­pound occurs preferentially at the para-configurated methoxy group. After perfusion with a mixture of (4-O-[14C] methyl) -and (3 -O -[14C] methyl) -3,4-dimethoxybenzoic acid under identical conditions the distribution of radioactivity of the named demethylation products was determined as 15:1. After perfusion with (4 -O -[14C] methyl) -3,4-dimethoxybenzaldehyde or (4-O -[14C] methyl)-3,4-di-methoxybenzoic acid alone, extracts of the perfusates revealed the expected radioactive 3-hydroxy-4-methoxybenzoic acid. In addition it was found, that the 4-hydroxy-3-methoxybenzoic acid pro­ duced contained small quantities of radioactivity. From these results it may be infered that an interconversion between 3-hydroxy-4-methoxy-and 4-hydroxy-3-methoxybenzoic acid has occured. The identification of the metabolites, separated by thin-layer chromatography, was performed by the reversed isotope dilution technique

STEROIDOGENESIS IN EQUINE TESTIS

1970 ◽  
Vol 64 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Ran Oh ◽  
Bun-ichi Tamaoki
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Isotope Dilution ◽  
Microsomal Fraction ◽  
Soluble Fraction ◽  
Enzyme System ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fluid ◽  
Layer Chromatography

ABSTRACT When the cell-free homogenates (supernatant fluid at 800 × g) and the supernatant fluid at 10 000 × g of equine testis were incubated with 4-14C-labelled progesterone and 17α-hydroxyprogesterone, radioactive androstenedione and testosterone were isolated as the metabolites. Oestrone was also produced by the same equine testicular preparation from the above mentioned C-21 steroids, androstenedione and dehydroepiandrosterone, while 17β-oestradiol was obtained from the testosterone which had been incubated as the substrate. In addition to the androgens and the oestrogens which were obtained as the metabolites, 19-hydroxyandrostenedione, 20α-dihydroprogesterone and androst-5-ene-3β,17β-diol were identified as the metabolites by thin layer chromatography, chemical derivation procedures, and isotope dilution methods in the course of recrystallization. The testicular 19-hydroxylase and aromatizing enzyme system were found to be concentrated intracellularly in the microsomal fraction (10 000-105 000 × g precipitate), while the 20α-hydroxysteroid dehydrogenase which preferably converted progesterone into 20α-dihydroprogesterone were exclusively found in the soluble fraction or the supernatant fluid at 105 000 × g. The role and mechanism of aromatization by equine testicular preparations are discussed in relation to those of the placental microsomal aromatase.

6.7-Dihydroxycumarin (Aesculetin) als Substrat der Catechol-O-Methyltransferase / 6,7-Dihydroxycoumarin (Aesculetin) as a Substrate for Catediol-O-methyltransferase

1976 ◽  
Vol 31 (5-6) ◽  
pp. 280-284 ◽  
Author(s):  
D. Müller-Enoch ◽  
E. Seidl ◽  
H. Thomas
Keyword(s):  
Thin Layer ◽  
Rat Liver ◽  
Isotope Dilution ◽  
Substrate Concentration ◽  
Dilution Technique ◽  
Isotope Dilution Technique ◽  
Fluorescent Compounds

6,7-Dihydroxycoumarin (Aesculetin) was found to be a substrate of rat liver Catechol-O-methylfransferase (COMT) (EC 2.1.1.6). Incubation of this substrate with S-Adenosyl-ʟ-[methyl-14C] methionine and/or S-Adenosyl- methionin-hydrogensulfate in the presence of COMT yields the highly fluorescent compounds 7-hydroxy-6-methoxycoumarin (Scopoletin) and 6-hydroxy-7-methoxycoumarin (Isoscopoletin) in the ration of about 2:1. The O-methylated products obtained from Aesculetin were identified after separation by thin layer chromatographpy mainly by the reversed isotope dilution technique. The fluorescence of the isolated methylethers was proportional to concentration within the range from 10-5 - 10-7M. A reciprocal plot of activity versus substrate concentration gives a Km of 1 × 10-6 M.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR).Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS.Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC–MS. Conclusions With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

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