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The subceflular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chhoroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADPGlucose pyropbosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosyhse) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction Chromatography of a chloroplast extract on diethylaminoethyl-ceilulose resolves the R-and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amyopectin as a substrate indicates an endolytic activity but displays properties unlike the typical a-amylase as isolated from endosperm tissue.

Abstract. The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 650 for 5 minutes in the presence of 0.02 M phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fruotose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents.The AO,5 (conc of activator required to give 50 %o of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 X 10-6 M while for the sorghum enzyme it was 3.7 X 10-4 M. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The 10.5 (conc of inhibitor that gave 50 % inhibition of activity for the various leaf enzymes varied from 2 X 10-M (barley) to 1.9 X 10-4 M (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.The hiosynithesis of the a-1,4 gluco,side linkage of starch from UDPglucose3 and ADPglucose in many pliawt exltracts have been the subject of many reporits (1, 2, 5, 8-16, 29--36) since the initiall obseirvatilons by Lelloir'is group (7,26,38). In alil extradts the rate of transfer of glucose from ADPglucose (reacltion 1) to the polysaicdharide prime,r was many-fold fa!ster than the rate of transfer from uridine dipho,sphalte gluco,se (UDPglucose) (re,action 2).

An enzyme catalyzing the synthesis of adenosine diphosphate (ADP) glucose from adenosine triphosphate (ATP) and -glucose 1-phosphate has been partially purified from Escherichia coli B. It was found that ADP glucose pyrophosphorylase activity could be stimulated by a number of glycolytic intermediates.

Soluble ADPglucose-az-glucan 4-a-glucosvltransferase (starch synthetase), ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase were assayed in extracts from developing kernels of maize (Zea mays). Normal, waxy and amylose-extender maize at stages of development ranging from 8 days to 28 days after pollination were studied. Shrunken-4 maize at the 22-day stage was also studied. There is adequate activity of both ADPglucose pyrophosphorylase and starch synthetase at all stages of development to account for the synthesis of starch. Thus all starch could be synthesized via the ADPglucose pathway. High levels of UDPglucose pyrophosphorylase and of phosphorylase activities were also found at all stages of development. The possible role of phosphorylase in starch synthesis could not be discounted. The levels of phosphorylase, ADPglucose pyrophosphorylase, starch svnthetase, and UDPglucose pyrophosphorylase activities in shrunken-4 kernels were about 20 to 40% of that found in normal maize kernels. It appears that the mutation in shrunken-4 affects the activities of more than one enzyme. The defective starch svnthesis seen in this mutant could be due to the low activities of ADPglucose pyrophosphorylase and starch synthetase rather than the low activity of phosphorylase.Biosynthesis of a-1 ,4-glucosidic linkages of starch in higher plants is generally considered to be catalyzed by ADPglucosea-,4-glucan 4-a-glucosyltransferase (starch synthetase) (13). It has recently been shown that some forms of this enzyme extracted from spinach, maize, and potato can synthesize a-1, 4-glucosidic linkages in the absence of added primer (7,11,12 Carbohydrate Determinations. To 0.5 g of frozen kernels were added 5 ml of 75% (v/v) ethanol. The kernels were thawed and ground in the ethanol and then heated for 20 min in a boiling H20 bath. After cooling, the suspension was centrifuged at 10,000g for 10 min. The supernatant fluid was decanted, and the starch precipitate was extracted a second time as above. The supernatant fluids were combined, evaporated to dryness, and dissolved in 1 ml of H20. This solution was used for analyses of reducing sugars (10), total soluble sugars (6), and sucrose (6).

The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained: glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity. One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000. The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000. Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction. The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3 kcal.

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 ...

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