Pyruvoyl Tetrahydropterin Synthetase

6-Pyrovoyl tetrahydropterin synthetase catalyzes the second step in the conversion of guanosine triphosphate into tetrahydrobiopterin. The substrate, 4,8-dihydroneopterin triphosphate, is converted to 6-pyruvoyl tetrahydropterin. 

Sepiapterin reductase is included in the assay to allow the product to proceed to tetrahydrobiopterin, which is then oxidized to biopterin, which is detected by fluorescence. 

The neopterin and biopterin derivatives were separated by chromatography on a LiChrosorb RP-18 column (4 mm x 250 mm, 7 /Am). The column was eluted at a flow rate of 0.8 mL/min with 0.015 M potassium phosphate buffer (pH 6.0). To elute biopterin, a pulse of 0.1 mL of 0.6 M potassium phosphate buffer (pH 6.8) was used. Biopterin was measured fluorimetrically by using excitation and emission wavelengths of 353 and 438 nm, respectively. 

The standard reaction mixture was composed of 5 /aL of Tris-HCl (pH 7.4), 5 /aL of 40 mM NADPH, 5 /aL of sepiapterin reductase (activity of 400 nmol/min/mL), and 65 /aL of cell extract (10-200 /Ag of protein). The reaction was started by the addition of 20 /aL of 0.4 mM 7,8-dihydroneopterin triphosphate. After 30 to 90 minutes of incubation in the dark at 37°C, the reaction was terminated by the addition of 50 /aL of a mixture of 0.2 M HC1 and 0.02 M KI-I2 (11, v/v). The resulting mixture was incubated for 1 hour in the dark to allow oxidation of tetrahydrobiopterin to biopterin. Excess iodine was destroyed by the addition of 50 /aL of 0.02 M ascorbic add. An aliquot of the mixture was applied to a solid phase cartridge (SCX from Analytichem) that had been preequilibrated with 0.1 M H3PO4. The sample was forced through the cartridge with air pressure. The cartridge was then washed with 0.5 mL of 0.1 M H3PO4. The eluates were used for HPLC analysis. Assays were linear with up to 150 fig of cellular protein and 90 minutes of incubation. 

Enzyme activity was measured in cell-free extracts of normal human fibroblasts and the human transitional cell bladder cardnoma line T-24. 

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Figure 9.150 HPLC profiles of incubation mixtures for 6-pyruvoyl tetrahydropterin synthetase assays with extracts of (A) T 24 cells, (B) human dermal fibroblasts, and (C) a reagent control. Amounts of 56 fig (A) and 60 fig (B) of cellular protein or phosphate-buffered saline (C) were used in the assay. Incubation time was 70 minutes at 37°C. A 100 fiL aliquot of the incubation mixture was used. Fluorescence detection was at an excitation wavelength of 353 nm and an emission wavelength of 438 nm. Peaks 1, neopterin 2, biopterin. (From Warner et al., 1991.)...

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See also in sourсe #XX -- [ , ]

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