FBPase assay

Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity was determined by monitoring the formation of fructose-6-phosphate (F6P) using a coupled spectrophotometric assay. The specific activity of rFBP1 was determined in 1 ml reactions containing 50 mM TES pH 7.4, 0.2 mM NADP⁺, 0.1 M KCl, 0.05 mM EDTA, 2 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.05 % (w/v) BSA, 2 mM 2-mercaptoethanol, 0.8 U/ml phosphoglucose isomerase and 0.5 U/ml glucose-6-phosphate dehydrogenase. Reactions were started by the addition of 35 μM F-1,6-P₂ and A₃₄₀ recorded in a Cary 100 spectrophotometer at 30°C. Reaction rates were calculated from the linear phase assuming ε(NADPH) = 6.22 mM^-1.cm^-1. 1 U is defined as 1 μmol F6P formed per min at 30°C. Where appropriate, 1 U/ml AMP deaminase (purified from chicken muscle using P11 phosphocellulose essentially as described by Smiley⁴) was included to remove contaminating AMP from NADP⁺ as described by Han⁵. The activity ratio at pH 7.2/9.4 was determined under similar conditions in reactions buffered with 50 mM bis-tris propane at the appropriate pH. Other kinetic properties were determined in a 96 well format in non-binding black microplates (Greiner #655900), where the quantity of NADP⁺ was reduced to 0.15 mM and reactions were monitored by the increase in fluorescence (λ_ex = 345 nm, λ_em = 465 nm) calibrated by the addition of 5 nmol F6P. K_m(F-1,6-P₂) was determined at 2 mM Mg²⁺ and fitted to equation 1:

Where: V = initial velocity, V_m = maximum velocity, [S] = F-1,6-P₂ conc., K_m = Michaelis constant for S, K_s = apparent substrate inhibition constant and b = factor determining maximum activity at high [S].

K_m(Mg²⁺) was determined at 35 μM F-1,6-P₂ and fitted to equation 2:

Where: V = initial velocity, V_m = maximum velocity, [S] = Mg²⁺ conc., K_m = Michaelis constant for S and h = hill coefficient.

IC₅₀ for inhibitory compounds was determined at 2 mM Mg²⁺, 35 μM F-1,6-P₂ and fitted to equation 3:

Where: V = initial velocity, V₀ = maximum velocity in the absence of inhibitor, I = conc. inhibitor, I_0.5 = conc. of inhibitor that gives 50 % inhibition and h = hill coefficient.

Stock solutions of F-1,6-P₂ were standardized by enzymatic assay in reactions containing 50 mM imidazole pH 7, 0.15 mM NADH, 0.02 U/ml aldolase, 1.2 U/ml triosephosphate isomerase and 0.16 U/ml glycerol-3-phosphate dehydrogenase. Stock solutions of AMP, 5’-inosine monophosphate (IMP) and 5’-AICAR monophosphate (ZMP) were prepared in 20 mM TES pH 7.4, neutralized with NaOH and standardized by UV absorbance in 0.1 M phosphate pH 7 at A₂₅₉ (ε_AMP = 15.4 mM^-1.cm^-1), A₂₄₉ (ε_IMP = 12 mM^-1.cm^-1) and A₂₆₅ (ε_ZMP = 12.5 mM^-1.cm^-1) respectively. F-2,6-P₂ is difficult to obtain commercially and the crude product available from Toronto Research Chemicals was solubilized in 10 mM NaOH and standardized in reactions containing 50 mM HEPES pH 7.1, 5 mM MgCl₂, 0.1 mM EDTA, 0.15 mM NADP⁺ by sequential addition of 0.1 U/ml glucose-6-phosphate dehydrogenase, 0.2 U/ml phosphoglucose isomerase and 0.25 U/ml GST-FBPase-2 P251-N440 while recording the increase in A₃₄₀.