Purification and Properties of Spinach Leaf Debranching Enzyme

Abstract

Starch debranching enzyme was purified from intact spinach (Spinacia okracea L. cv Vital) chloroplasts and from a spinach leaf extract using affinity chromatography on Sepharose 6B-bound cycloheptaamylose (Schardinger,B-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 molecular weight of the native protein (110,000 daltons) was not changed by treatment with sodium dodecyl sulfate. Only one spinach leaf debranching enzyme band could be detected after electrophoresis of a leaf extract on amylopectin-containing polyacrylamide gel, the retardation factor of 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 units per milligram protein with pullulan and 22 units per milligram protein with amylopectin. Cycloheptnamylose is a potent competitive inhibitor of spinach leaf debranching enzyme. The pH optimum of the enzyme was found to be 5.5. The purified enzyme is rather unstable at both 200 and 0°C. Part of the activity lost under storage or at a suboptimal pH could immediately be restored by the addition of thiols. The reactivatable protein, being of the same molecular weight as the native enzyme, exhibited a somewhat altered electrophoretic mobility resulting in one or two minor bands on a zymogram.In contrast to the enzymes of assimilatory starch formation, which are located exclusively in the chloroplast, the enzymes of starch degradation have been found in the cytosol as well as in the chloroplasts (1 1, 14). Their activities in the former compartment usually exceed those in the plastids by far (13). MATERIALS AND METHODSPreparation of Leaf Extract. Deribbed spinach leaves (Spinacia oleracea L. cv Vital; 820 g) were cut into small pieces and homogenized in 1,500 ml 50 mm sodium acetate buffer (pH 6.0) with a Multimix. The homogenate was then centrifuged at 16,300g for 30 min at 0°C. Solid (NH4)2 S04 was added to the supematant and the precipitate which formed between 40 and 50% saturation was used for further purification of the enzyme. The precipitated protein was dissolved in 20 to 30 ml of acetate buffer (as described above) and dialyzed for 12 h against bidistilled H20. Prior to affinity chromatography, the solution was freed from precipitated protein by another centrifugation step (45,000g for 30 min).Preparation of Chloroplast Extract. Intact chloroplasts were isolated from spinach leaves using the Mes and Hepes buffers 'A' and 'B' as described by Jensen and Bassham (7). The whole procedure was performed at 0°C. Deribbed spinach leaves (250 g) were homogenized in buffer A for 30 s with an Ultraturrax. The homogenate was filtered through eight layers of gauze and centrifuged for 2 min at 2,600g. The precipitate was suspended in 120 ml buffer B and cells and cell debris were removed by centrifugation at 30g (twice). From the sup...

Characterization and Subcellular Localization of Debranching Enzyme and Endoamylase from Leaves of Sugar Beet

“…These results support the view that DTT affects enzyme activity by changing the conformations of the enzyme rather than simply protecting it from denaturation (18).…”

“…For instance, the role of starch-debranching enzyme in green leaves has been assumed to be the same as that ofthe enzyme in cereal grains ( 19). Although a debranching enzyme from leaves of spinach has been purified and partially characterized (18), it is not known whether this enzyme has the ability to initiate the breakdown of grains of transitory starch.…”

Independent Regulatory Aspects and Posttranslational Modifications of Two β-Amylases of Rye

“…The effectiveness of this immunoelimination is shown in Figure 2A. The same IEF diagrams were obtained with fresh and stored extracts indicating that none of the heterogeneity observed was due to storage artifacts as was the case for the unstable debranching enzyme extracted from spinach leaves (13).…”

Partial Purification and Characterization of the Major Endoamylase of Mature Pea Leaves

“…Preincubation of highly diluted preparations of the purified endoamylase (100-fold dilutions of the final concentrate described in Table I) without substrate for 10 to 30 min as described in Figure SB always resulted in the loss of a considerable proportion of the activity determined when the standard assay was (14) was thus evident with these compounds. The effects of three other groups of substances potentially inhibitory to amylolytic activity are compiled in Table III.…”