title: 6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human cells using high-performance liquid chromatography with fluorescence detection of biopterin.
abstract: An assay for 6-pyruvoyl tetrahydropterin synthase, the second enzyme in the conversion of guanosine triphosphate into tetrahydrobiopterin, has been developed. Cell extracts were incubated with enzymatically prepared dihydroneopterin triphosphate (80 microM) in the presence of Mg2+ (12 mM), excess sepiapterin reductase (EC 1.1.1.153) (2 nmol/min) and NADPH (2 mM). 6-Pyruvoyl tetrahydropterin, the product of the reaction, was thus converted into tetrahydrobiopterin. After oxidation of the reduced biopterin derivatives in acidic iodine solution, biopterin was enriched and separated from the abundant neopterin phosphates by solid-phase extraction on a strong cation exchanger. Biopterin was then directly eluted on a reversed-phase liquid chromatographic column and detected fluorimetrically using excitation at 353 nm and emission at 438 nm. The biopterin concentrations formed by the coupled enzyme reaction increased linearly with incubation times up to 90 min. The assay allows the quantification of 6-pyruvoyl tetrahydropterin synthase in cultured human cells.
authors.affiliation: Institute for Medical Chemistry and Biochemistry, University of Innsbruck, Austria.
authors.forename: E R
authors.initials: ER
authors.lastname: Werner
authors.forename: G
authors.initials: G
authors.lastname: Werner-Felmayer
authors.forename: D
authors.initials: D
authors.lastname: Fuchs
authors.forename: A
authors.initials: A
authors.lastname: Hausen
authors.forename: G
authors.initials: G
authors.lastname: Reibnegger
authors.forename: G
authors.initials: G
authors.lastname: Wels
authors.forename: J J
authors.initials: JJ
authors.lastname: Yim
authors.forename: W
authors.initials: W
authors.lastname: Pfleiderer
authors.forename: H
authors.initials: H
authors.lastname: Wachter
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001708:Biopterins / Q000032:analysis*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D019761:Phosphorus-Oxygen Lyases; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
chemical_list: D001708:Biopterins; D000429:Alcohol Oxidoreductases; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase