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

Abstract

Sugar beet leaves (Beta vulgaris L.) contained up to five endoamylases, two exoamylases, and a single debranching enzyme. Four of the endoamylases and the debranching enzyme were present in the chloroplast. The chloroplastic starch-debranching enzyme and an apoplastic endoamylase were copurified from mature leaves of sugar beet by 35 to 50% ammonium sulfate precipitation and chromatography on diethylaminoethylSephacryl, jB-cyclodextrin Sepharose 6B, and Sephadex G-150. The debranching enzyme, which was purified to homogeneity, had a molecular mass of 100 kilodaltons and a pH optimum of 5.5. It showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with native starch grains isolated from sugar beet leaves. The endoamylase, which was partially purified, had a molecular mass of 43,000 kilodaltons, a pH optimum of 6.5, required calcium for activity and thermal stability, and showed an ability to hydrolyze nafive starch grains.Sugar beet leaves sustain export of assimilated carbon to sink tissues by accumulating a high level of starch during the day and degrading this starch at night. Degradation of starch appears to be a regulated process because it responds to day length (6) and the rate of starch synthesis during the previous light period (17). Furthermore, the rate of starch degradation in the light responds to photosynthesis rate (1 1 endoamylase activity is outside the chloroplast and is present even in the apoplast (3) needs to be considered when evaluating its physiological role in plant leaves. Likewise, knowledge of the roles of other chloroplastic starch hydrolytic enzymes is scant. 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.To learn more about the process of starch degradation and its regulation, we initiated a study of possible roles of the various amylases present in sugar beet leaves. Here, we describe the purification, biochemical properties, and subcellular localization of two of these enzymes. MATERIALS AND METHODS Plant MaterialSugar beet (Beta vulgaris L. cv Klein multigerm) plants were grown as described previously (10). Enzyme PurificationAbout 300 g of leaves from 3-to 4-week-old plants was washed, cut into small pieces, and homogenized in 1.2 L of ice-cold extraction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mm 2-mercaptoethanol, 3 mm CaCl2, and 0.5 mM PMSF. The homogenate was filtered through two layers of cheesecloth and then clarified by centrifugation at 10,400g for 20 min at 4°C. Solid (NH4)2SO4 was then added to the supernatant with stirring, and the precipitate formed between 35 and 50% saturation was collected and dialyzed overnight at 4°C against 2 L of buffer A (50 mm Tris-HCl [pH 7.2], 10 mM 2-merc...

Characterization of Starch-Debranching Enzymes in Pea Embryos

“…The pea enzyme hydrolyzes pullulan more readily than amylopectin and displays little or no detectable activity with glycogen. This order of substrate preference is the same as that of the enzymes from, for example, spinach chloroplasts (Okita and Preiss, 1980;Ludwig et al, 1984), sugar beet leaves (Li et al, 1992), mature broad bean seeds (Gordon et al, 1975), and developing rice endosperm (Nakamura et al, 1996a). The properties of our pea enzyme are also very similar to those of a limit-dextrinase purified by ammonium sulfate and acetone fractionation and cyclohexaamylose chromatography from imbibed pea seeds (Yellowlees, 1980).…”

“…Cell-fractionation studies showed that approximately onehalf of the activity of spinach (Okita et al, 1979), sugar beet (Li et al, 1992), and broad bean (Ghiena et al, 1993) leaves is chloroplastic. Studies of other organs have for the most part identified only one activity, for example in germinating barley (Sissons et al, 1992;MacGregor et al, 1994); developing and germinating rice endosperm (Iwaki and Fuwa, 1981;Nakamura et al, 1996a); maize endosperm (Doehlert and Knutson, 1991); and broad bean seeds (Gordon et al, 1975).…”

“…Limit-dextrinase activity occurs in leaves, embryos, and endosperms of several species (Nakamura, 1996). There is evidence that the enzyme in developing rice endosperm is plastidial (Nakamura et al, 1996a), and both plastidial and extraplastidial activities have been found in leaves (Okita et al, 1979;Li et al, 1992;Ghiena et al, 1993), but the location of the activity and the number of different gene products involved in other organs are not known. Isoamylase activity has been reported only from maize endosperm, in which it is plastidial (Doehlert and Knutson, 1991;Yu et al, 1998), and potato tuber, in which its intracellular location is not known (Ishizaki et al, 1983).…”

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

“…However, a-amylase activity associated with chloroplasts could originate from extrachloroplastic contamination. Recently, the chloroplastic localization of an a-amylase has been reported in sugar beet by Li et al (1992).…”

“…PEP carboxylase (EC 4.1.1.3 1) and NAD-malate dehydrogenase (EC 1.1.1.37) were assayed using methods similar to those described by Li et al (1992). Final NAD-malate dehydrogenase assay concentrations were 50 m~ Tris-HC1 (pH 7.5), 0.2 mM NADH, and 0.5 mM oxaloacetic acid.…”