2 14c iaa

IAGlc synthase activity was determined in a total volume of 8 μL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA, 0.016 μCi [2′-¹⁴C]IAA (55 mCi mmol⁻¹, Hartmann Analytic GmBH, Braunschweig, Germany) and 2.5 mM MgCl₂ with 3 μL of the enzyme preparation. The reaction was stopped after 15 min incubation at 30 °C by drying 4 μL of aliquots on a Silica Gel F₂₆₀ TLC plate (Merck, Darmstadt, Germany). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5/3/1/1, v/v/v/v) as a solvent. The indole ring compounds were visualized by staining the plate with the Van Urk–Salkowski reagent [44 (link)]. Bands identified as IAGlc were excised and placed in a vial with 2 mL EcoLite (+) scintillation fluid (ICN). The radioactivity level was measured using a Wallac 1409 liquid scintillation counter (Turku, Finland).

The effect of UDPG concentration on the IAGlc synthase activity was analyzed in a total volume of 8 μL containing 25 mM HEPES buffer, pH 7.4, 3 mM IAA and 0.016 μCi [2′-¹⁴C]IAA (55 mCi mmol⁻¹, Hartmann Analytic GmBH, Germany) with 3 μL of the enzyme preparation with different UDPG concentrations (0.5–20 mM). The reaction was stopped after 15 min incubation at 30 °C by drying 4 μL of aliquots on a Silica Gel F₂₆₀ TLC plate (Merck). The rest of the assay was performed as described above. The V_max and K_M parameters were calculated using a Hanes–Woolf plot ([S]/v = f_([S])) and verified by the Michaelis–Menten equation using SigmaPlot 11.0 (Systat Software Inc, San Jose, California, USA).

Enzymatic activity of IAInos synthase towards IAInos biosynthesis was determined in a total volume of 8 μL containing 25.2 mM HEPES buffer, pH 7.4, 12 mM UDPG, 6.4 mM IAA, 592 Bq [2’-¹⁴C]IAA (2.035 GBq mmol⁻¹; Hartmann Analytic GmBH), 15 mM myo-inositol, 18 mM D-gluconic acid lactone, 4 mM MgCl₂ and 3 μU of recombinant IAGlc synthase, with 3 μL of the supernatant fluid from tissue homogenates. The reaction was stopped after 30 min incubation at 30 °C by drying 4 μL of aliquots on Silica Gel F₂₆₀ TLC plate (Merck). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5: 3: 1: 1, by vol.) as a solvent. For indole compounds visualization, the plate was stained with van Urk-Salkowski reagent (Ehmann 1977 (link)). Bands corresponding to IAInos were excised and placed in a vial with 2 mL EcoLite ( +) scintillation fluid (MP Biomedicals). Radioactivity level was measured in Wallac 1409 liquid scintillation counter (Wallac Oy).