Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves.

The non-chloroplastic α-glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.