GTPCH activity

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

Additional freeze-thawing of tissue or fibroblast lysates may result in reduced GTPCH activity, and it is thus recommended to assay activity always from freshly lysed material. Alternatively, lysates may be kept for 1-2 days at -80°C (this may not be the case for PTPS, SR, and DHPR assays, as extracts can be kept at -80°C for a longer period without loosing activity). 

GTPCH activity measurements are not available in external control programs and can only be compared by exchanging results from other laboratories (for examples see www.bh4.org). Control biopterin and neopterin samples are commercially available from Dr. Schircks Laboratories, Jona, Switzerland (www.schircks.com). We recommend using internal controls (e.g., normal control fibroblasts in each enzyme... 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. GTPCH activity is assayed by measuring the neopterin produced upon enzymatic incubation at 37°C for 60 min in a final volume of 0.1 ml in the dark (due to light sensitivity of pterins), followed by chemical oxidation and dephosphorylation. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the neopterin that was present in the lysate, and a blank reaction without cell lysate to detect the neopterin that is generated from the incubation (substrate) buffer. The sum of both blanks is later subtracted from the value of the incubation reaction to determine the enzymatically produced neopterin. 

GTPCH activity is determined as pU/mg protein. One unit of GTPCH produces 1 pmol neopterin/min at 37°C. The results from the neopterin (N) determination by HPLC are given in nmol/1. 

A confluent fibroblast cell monolayer in 78-cm2 plates, cultured in fresh DMEM (see section 6.1.4.1, subheading Specimen ), is incubated with recombinant human INF-y (2.5 pi per 5 ml DMEM 1250 U) and TNF-a (2.5 pi per 5 ml DMEM 500 U). After stimulation for 24 h, cells are harvested by trypsinization, washed with PBS, and immediately lysed for neopterin and biopterin measurements and for GTPCH activity assay. 

GTPCH, FTPS, and SR are required and sufficient to carry out the proper stereospecific reaction to BH4 (45). With the crystallographic structures (Figure 13), including the characteristics of the active centers of all three enzymes, the essential information for the interpretation of the reaction mechanism is available. Moreover, NMR studies on the reaction mechanisms of all three enzymes revealed the details of the hydrogen transfer process and the stereochemical course of the reactions." ... 

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