Purification and Properties of Spinach Leaf Debranching Enzyme

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Abstract
Starch debranching enzyme was purified from intact spinach chloroplasts and from a spinach leaf extract using affinity chromatography on Sepharose 6B-bound cycloheptaamylose (Schardinger .beta.-dextrin). The enzyme from both sources was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spinach leaf debranching enzyme appears to consist of a single polypeptide chain, since the MW of the native protein (110,000 daltons) was not changed by treatment with sodium dodecyl sulfate. Only 1 spinach leaf debranching enzyme band could be detected after electrophoresis of a leaf extract on amylopectin-containing polyacrylamide gel, the retardation factor which coincided with that of the single band seen with the chloroplast enzyme. The purified enzyme exhibited strong pullulanase activity, the specific activity being 69 U/mg protein with pullulan and 22 U/mg protein with amylopectin. Cycloheptaamylose is a potent competitive inhibitor of spinach leaf debranching enzyme. The pH optimum of the enzyme was 5.5. The purified enzyme is rather unstable at both 20.degree. and 0.degree. C. Part of the activity lost under storage or at a suboptimal pH could immediately be restored by the addition of thiols. The reactivable protein, being of the same MW as the native enzyme, exhibited a somewhat altered electrophoretic mobility resulting in 1 or 2 minor bands on a zymogram.