Potato amylopectin

The leaf samples (300 mg) harvested after 8 h light or 4 h dark were ground in liquid nitrogen, homogenized in 1 mL of soluble protein extraction buffer (50 mM Hepes pH 7.5, 2 mM EDTA, and 10% glycerol), and then incubated for 5 min on ice. The supernatant was obtained after 10 min of centrifugation at 14,000× g at 4 °C and soluble protein concentration was measured by Bradford assay (Bio-Rad, Hercules, CA, USA) while using bovine serum albumin as a standard. Protein extracts (35 µg) were incubated in the presence or absence of 40 mM DTT or 200 µM CuCl₂ for 2 h on ice in the dark and loaded on native PAGE gel (7.5% acrylamide) containing 0.2% (w/v) potato amylopectin (Sigma-Aldrich, Saint Louis, MO, USA) as substrate in the separating gel. After migration (under native condition for 4 h at 4 °C at 15 V.cm⁻¹), the gels were incubated overnight at room temperature in 100 mM Tris-HCl pH 7, 1 mM MgCl₂, and 1 mM CaCl₂ containing buffer. Activities were revealed by iodine staining (Lugol solution; Sigma-Aldrich, Saint Louis, MO, USA).

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.