Quantifying Potato Amylopectin Phosphate Release

Release of phosphate from potato amylopectin was determined as done previously (23 (link), 24 (link), 42 (link), 93 (link)) with the following modifications. Briefly, phosphate release was monitored using the PiColorLock Phosphate Detection Reagent (Novus Biologicals), a malachite green–based assay. For time course assays, 5 nM recombinant protein was incubated with 90 μg solubilized potato amylopectin (Sigma-Aldrich), supplied as a powder; solubilized at a stock concentration of 5 mg/ml using alcohol/alkaline method (also referred to as the “Roach method” in Ref. (93 (link))) in phosphatase buffer in a final volume of 80 μl at pH 6.5. For TgLaforin kinetic characterization, 5 nM TgLaforin was incubated with varying amylopectin concentrations for 10 min. All reactions were terminated by addition of 20 μl (0.25 initial reaction volume) of the PiColorLock Gold solution containing Accelerator in a 100:1 ratio of Gold solution to the accelerator. After 5 min, 8 μl stabilizer solution (0.1 initial reaction volume) was added, and reaction was allowed to develop for 30 min at r.t. before the absorbance of each reaction was measured at 635 nm using a Synergy HTX Multi-Mode Reader (BioTek). Absorbance was converted to pmol P_i release using a P_i absorbance standard curve. Data points are presented as the mean of three independent replicates, each consisting of three technical replicates.