Lichrosphere

Recombinant human guanylate cyclases 1 and 2 (GC1 and GC2, respectively) were expressed in permanent HEK293 cells after transfection with PEI and 10-day selection with geneticin [69 (link)]. After cell lysis, cell membranes containing GCs were resuspended in 50 mM HEPES pH 7.4, 50 mM KCl, 20 mM NaCl, 1 mM DTT buffer and incubated with 5 µM GCAP1 variants for 10 min at 30 °C in the presence of 1 mM Mg²⁺ and either 2 mM EGTA (free Ca²⁺ < 19 nM) or ~30 µM Ca²⁺ [26 (link)]. Enzymatic reactions were carried out in 30 mM MOPS/KOH pH 7.2, 60 mM KCl, 4 mM NaCl, 1 mM GTP, 3.5 mM MgCl₂, 0.3 mM ATP, 0.16 mM Zaprinast buffer and blocked with the addition of 50 mM EDTA and boiling. Samples were then centrifuged for 20 min at ~18,000× g at 4 °C; finally, the supernatant was loaded on a C18-RP column (Lichrosphere, Merck) to quantify cGMP production. Control experiments were performed in the absence of GCAP1 to measure basal cGMP synthesis for each of the independent replicas. The production of cGMP at low and high Ca²⁺-concentration in the presence of GCAP1 variants was subjected to two-tailed t-tests, where the null hypothesis was represented by equal averages between different conditions. All differences were statistically significant (p-values < 0.016).

Phenylalanine hydroxylase (PAH) activity was assessed as described previously [44 (link)]. Briefly, a rat PAH maltose fusion protein was derived from rat liver amplified PAH cDNA cloned in pmalC2 (NEB) and expressed in E. coli. The fusion protein was purified using an amylose column and elution with maltose. In order to assess PAH activity, 60 µg/mL recombinant rat PAH were preincubated with 200 µM phenylalanine for 10 min at 37 °C, and the reaction was started by adding a mixture of 24, 75 or 240 µM tetrahydrobiopterin (BH4) and 104 units/mL catalase (Serva, Heidelberg, Germany). After incubation for 20 min at 37 °C, the reaction was stopped by adding 10 µL of 1 M HCl. Ten microliters were then injected onto an RP-18 column (125 × 4 mm, Lichrosphere, 5 µm particle size; Merck) and eluted with 75 mM KH₂PO₄ buffer containing 20% (v/v) acetonitrile, 10% (v/v) methanol, 0.44 g/L SDS and 1.5 µM EDTA, pH 3.1 (adjusted with 1 M trichloroacetic acid), at a flowrate of 0.8 mL/min. Tyrosine was detected by fluorescence (excitation 285 nm, emission 325 nm) with a detection limit of 2.5 pmol (0.25 µM). Effects of compound 6 (2’N-dimethyl-6,7-dimethyl tetrahydropterin) were determined in 3 independent experiments with 0, 3, 10, 100, 300 and 1000 µM inhibitor (dissolved in DMSO) in the presence of 24, 75 and 240 µM BH4.