Characterization of the Spinach Leaf Phosphorylases

Abstract

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The Little is known about the primary steps involved in chloroplastic starch degradation. Recent investigations on starch degradation in isolated spinach chloroplasts have indicated that both maltose and 3-P-glycerate are end products (8,13,20). This suggests that in spinach chloroplasts both amylolytic and phosphorolytic cleavages are occurring. In contrast, negligible amylolytic activity is found in pea chloroplast extracts, and starch degradation appears to occur solely via phosphorolysis (14, 24).Thus far there is little available information on the isolation and characterization of the starch degradative enzymes in spinach leaf. Recent studies indicate that multiple forms of phosphorylase do exist in spinach leaf (3,19,23 Preparation of Debranched Amylopectin. Two g amylopectin in 50 ml of 20 mm citrate buffer (pH 5.0) was incubated with 50 ,ug pullulanase (Boehringer Mannheim, 10 IU/mg) at 37 C for 21 h. The debranched amylopectin was then heated at 100 C for 5 min and clarified by centrifugation at 15,000g for 10 min. The mixture was then diluted with H20 to a final concentration of 10 mg/ml. This preparation contains two maltodextrin fractions; one with a chain length range between 12 to 42 glucosyl residues and the other with chain lengths greater than 49 glucosyl residues (7).Preparation of Partially Hydrolyzed Amylose. Potato amylose type I, 4.5 g, was suspended in 225 ml of 0.1 N HCI and boiled in a water bath for 60 min. The solution was cooled and neutralized with 10 N NaOH to pH 7.0.Sucrose Density Gradient Ultracentrifugation of Phosphorylase. Sucrose density ultracentrifugation was done according to the procedure of Martin and Ames (16). Lactate dehydrogenase (mol wt, 140,000) and pyruvate kinase (mol wt, 237,000) were used as marker enzymes.Preparation of the Glycogen Affinity Resin. An affinity column with glycogen attached to Sepharose 6B was prepared according to the method of Vretblad (27). Epoxy-activated Sepharose 6B (Pharmacia), 15 g, was suspended in 94 ml deionized H20 and washed with 1,500 ml H20 for I h on a coarse glass filter. The gel was washed with 94 ml of 0.1 M NaOH and then transferred to 45 ml of a 0.1 M NaOH solution containing 25 mg ml-' of rabbit liver glycogen. The suspension was incubated at 45 C for 16 h with gentle shaking. The gel was then washed, successively, with 375 ml deionized H20, 750 ml of 25 mg/ml glucose solution, 375 ml deionized H20, 750 ml of 25 mg/ml glucose solution, 375 ml deionized H20, 375 ml of 0.1 M borate buffer (pH 8.0) containing 0.5 M KCI, 375 ml of 0.1 M Na-acetate buffer (pH 4.0) containing 0.5 M KCI, and finally with 375 ml deionized H20. The gel ...

Subcellular Localization and Characterization of Amylases in Arabidopsis Leaf

“…), debranching enzyme (EC 3.2.1.10), phosphorylase (EC 2.4.1.1. ), and D-enzyme (EC 2.4.1.25) (14,15,19). Amylase and phosphorylase activities constitute the major degradative activity in the chloroplast.…”

Purification and Properties of Spinach Leaf Debranching Enzyme

“…It has been demonstrated that amylolysis can act as pacemaker for the final phosphorolytic degradation of assimilatory starch (1). Whereas the phosphorylases have been purified to homogeneity (14,16), pure chloroplastic amylases could not be obtained by the classical separation techniques.…”

Purification and Properties of Mesophyll and Bundle Sheath Cell α-Glucan Phosphorylases from Zea mays L.

“…How-(b) The substrate affinity of phosphorylase I and II is identical ever, the Km for glycogen was 15 times smaller for phosphorylase for phosphate and soluble starch and differs for glycogen 20 to I than for phosphorylase II. Maltose, maltotriose, and malto-300-fold in spinach leaves (17,28) and 15-fold in corn leaves tetraose were ineffective substrates for both phosphorylases (Ta-(this paper). Differences exist in the affinity for amylopectin ble III).…”

“…Phosphorylase I has a dimeric structure in the C3 plant spinach (17,22,27) and in the C4 plant corn (this paper). The same situation applies to the subunit structure of phosphorylase II in C3 plants (22,24 (2,3,16,23), regulatory enzymes of fructose 1,6-bisP/fructose 2,6-bisP/fructose 6-P metabolism (30), and enzymes of nitrate fixation and ammonia fixing enzymes (6,12).…”

“…They are part of two sets of isoenzymes in glycolysis, gluconeogenesis and the oxidative pentose phosphate cycle in the cytosol and in the plastids of plant cells, respectively (20). The cytosol and plastid phosphorylases of C3 plant leaves have many properties in common; however, they differ greatly in their substrate affinity for glycogen, in their effect on some maltodextrins (17,22,28), and in their immunological crossreactivity (5,29).…”