Purification of chloroplast alpha-1,4-glucan phosphorylase from spinach leaves by chromatography on Sepharose-bound starch.

Chloroplast alpha-1,4-glucan phosphorylase (EC 2.4.1.1) has been purified to homogeneity from spinach leaves as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purification procedure is composed of (NH4)2SO4 precipitation, ion-exchange chromatography, and chromatography on Sepharose-bound starch. In order to achieve binding of the chloroplast phosphorylase, a previously described Sepharose-glucan gel (Steup, M. Schächtele, C. and Latzko, E. (1980) Planta 148, 168-173) was modified by introducing hydrophobic groups in addition to the covalently bound starch. The chloroplast phosphorylase exhibited complete binding to this type of gel and could be eluted by a mixture of soluble glucan and NaCl. For the purified chloroplast phosphorylase, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and pyridoxal phosphate determination resulted in a molecular weight estimation of about 110,000 per monomer. The apparent molecular weight of the native enzyme, as determined by polyacrylamide density gradient electrophoresis and gel filtration on Sephadex G-200, was 200,000 and 220,000, respectively. The data indicate that the chloroplast phosphorylase is a dimer with a molecular weight higher than that of the non-chloroplast phosphorylase.