Purification and Characterization of α-Glucan Phosphorylase Isoform Pho 2 from Spinach Leaves.

ABSTRACT

α-Glucan phosphorylase is an important enzyme of carbohydrate metabolism. In spinach leaves, it has been reported in two multiple forms viz. Pho 2 (cytosolic) and Pho 1 (plastidial). Here, we extracted and purified Pho 2 form of α-glucan phosphorylase from spinach using salting out with ammonium sulfate, desalting using Sephadex G-25 chromatography, anion exchange chromatography using DEAE-Sepharose and gel filtration chromatography using Sepharose-4-B. The purified enzyme had a specific activity, 150 units/mg protein. There was 38% recovery and 652 fold purification after final Sepharose-4B chromatography. The purified enzyme showed a single protein band on SDS sodium dodecyl sulfate polyacrylamide gel electrophoresis having molecular weight 94,000±2000 daltons. The native molecular weight is found to be 188,000±3000 daltons as determined using gel filtration chromatography over Sephadex G-200. The Pho2 exhibited optimum pH at pH 6.0 with two half pH optima at pH 5.2 and pH 7.0. The optimum temperature of Pho2 is found to be 37ºC with two half temperature optima at 30ºC and 40ºC. The Km value of the enzyme for starch and glucose-1-phosphate is found to be 116 μg/mL and 0.55 mM, respectively.