PanK enzymatic assay.

PanK activity was assayed by determining ATP consumption and ADP formation in the reaction using reverse-phase high-performance liquid chromatography (HPLC) analysis. The standard assay was performed at 70°C for 30 min in a mixture (100 μl) consisting of 5 mM calcium d-pantothenate (Tokyo Chemical Industry, Tokyo, Japan), 5 mM ATP (Oriental Yeast, Tokyo, Japan), 10 mM MgCl₂, 60 mM NH₄Cl, 60 mM KCl, and a designated amount of the enzyme in 50 mM Tris-HCl buffer (pH 7.5). As an alternative substrate, d-pantetheine was prepared by reducing d-pantethine (Merck) with an equivalent molar amount of 1,4-dithiothreitol (Fujifilm Wako). d-Pantoate was prepared by hydrolyzing d-pantolactone (Tokyo Chemical Industry) in an equimolar concentration of an NaOH solution. Enzyme solutions were preincubated at 70°C for 2 min before adding the substrates. The reaction was stopped by adding 20 μl of 1 M HCl to the mixture. Samples were centrifuged at 20,400 × g at 4°C for 5 min, and the supernatants were subjected to HPLC analysis. The analysis was performed using a 250-by-4.6 Cosmosil 5C₁₈-AR-II column (Nacalai Tesque) with a binary gradient of 20 mM tert-butylamine (pH 6.8; adjusted by H₃PO₄) and 20 mM tert-butylamine (pH 6.8) (plus 10% [vol/vol] methanol) as solvents A and B, respectively, at a flow rate of 1 ml min⁻¹. The sample injection volume was set to 15 μl, and the UV-visible (UV-vis) detector was set at a wavelength of 254 nm. The initial condition (0% solvent B) was increased linearly to 100% solvent B over 10 min. After a 2-min hold at 100% solvent B, there was a linear decrease to 0% solvent B over 15 s, followed by a 5-min hold at the initial condition (0% solvent B).