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).
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