Liquid Chromatography–Tandem Mass Spectrometry Enzyme Assay for UDP-Galactose 4′-Epimerase: Use of Fragment Intensity Ratio in Differentiation of Structural Isomers

Li, Yijun; Huang, Xiaoping; Harmonay, Lauren; Liu, Ying; Kellogg, Mark D.; Fridovich‐Keil, Judith L.; Berry, Gerard T.

doi:10.1373/clinchem.2013.219931

Cited by 6 publications

(2 citation statements)

References 19 publications

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“…Gonads were added to a tube containing PBS buffer, 1% proteinase inhibitor cocktail (Sigma-Aldrich), and lysed by sonication at 4 °C. Protein measurement, galk1 assay, and LC-MS/MS were performed as previously described (Li et al 2014 ), except that 1 mg/mL ovarian lysate were added to the assay co-factors cocktail and incubated at 30 °C for 30 min. Briefly, galk1 assay consisted of 12.5 μL of 2 mM 13 C 6 -galactose, 12.5 μl of 0.8 mol/L MOPS buffer pH 7.8, 12.5 μL mixture of co-factors cocktail (30 mM ATP, 32 mM MgCl 2 and 32 mM NaF) with 25 μl of 6 mg/mL ovarian lysate.…”

Section: Methodsmentioning

confidence: 99%

“…Frozen tissues were added to a tube containing 0.5 mol/L glycine pH 8.5 (Sigma-Aldrich), 1% proteinase inhibitor cocktail (Sigma-Aldrich), and lysed by sonication at 4 °C. Protein measurement, gale assay, and LC-MS/MS settings were the same as previously described (Li et al 2014 ) except that 20 μl of 4 mg/ml of brain or 1 mg/ml ovarian lysate were added to the cocktail and incubated at 30 °C for 30 min. 13 C 3 -UDP-Glc (Sigma-Aldrich) was added as internal standard.…”

Section: Methodsmentioning

confidence: 99%

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Impaired fertility and motor function in a zebrafish model for classic galactosemia

Vanoevelen

Erven

Bierau

et al. 2017

J of Inher Metab Disea

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Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate galactose-1-phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-017-0071-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Impaired fertility and motor function in a zebrafish model for classic galactosemia

Vanoevelen

Erven

Bierau

et al. 2017

J of Inher Metab Disea

Self Cite

View full text Add to dashboard Cite

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KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates

Zhu

Sun

et al. 2017

Appl Microbiol Biotechnol

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Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k /K value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.

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A novel on-flow mass spectrometry-based dual enzyme assay

Seidl

Vilela

Lima

et al. 2019

Analytica Chimica Acta

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Liquid Chromatography–Tandem Mass Spectrometry Enzyme Assay for UDP-Galactose 4′-Epimerase: Use of Fragment Intensity Ratio in Differentiation of Structural Isomers

Cited by 6 publications

References 19 publications

Impaired fertility and motor function in a zebrafish model for classic galactosemia

Impaired fertility and motor function in a zebrafish model for classic galactosemia

KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates

A novel on-flow mass spectrometry-based dual enzyme assay

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