The De Novo Synthesis of Polysaccharide by Phosphorylase

Illingworth, Barbara; Brown, David H.; Cori, Carl F.

doi:10.1073/pnas.47.4.469

Cited by 73 publications

(16 citation statements)

References 2 publications

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“…Since starch transglucosylases require a primer to start starch synthesis, it has been suggested that phosphorylase is important in primer syntheesiis (11).…”

Section: Resultsmentioning

confidence: 99%

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Phosphorylases I and II of Maize Endosperm

1968

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A bstract. Two phosphorylases have been found in the endosperm of Zea mays. Phosphorylase I is found through all stages of endosperm development and seed germination investigated. The Phosphorylase (a-1,4-glucan: orthophosphate glucosyl,transferase) was first discovered in liver tissue (2) to catalyze a reversible reaction between glucose-1-phosphate (glucose-1-P) and glycogen.Glucose-i-P + Glycogen (G)n ;± Glycogen (G)n +l + Pi. Soon after this discovery, the same type of reaction was demonstrated in tissues of higher plants (8, 9

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“…Since starch transglucosylases require a primer to start starch synthesis, it has been suggested that phosphorylase is important in primer syntheesiis (11).…”

Section: Resultsmentioning

confidence: 99%

“…rema,ins a problem. Recently, studies on muscle phosphorylase and potato phosphorylase showed that the de novo synthesis of amylose can proceed in the complete absence of primer (11) which suggests that phosphorylase could be of special importance…”

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confidence: 99%

Phosphorylases I and II of Maize Endosperm

1968

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“…A similar result in developing pea seeds was observed by Turner (19). It should be pointed out that the activities of both UDPglucose pyrophosphorylase and phosphorylase, enzymes implicated in starch synthesis (8,(16)(17)(18), are about 20-to 500-fold in excess of the ADPglucose pyrophosphorylase and transferase levels in the developing endosperm. The activities found for these en- There is also a 6-to 10-fold increase in the phosphorylase level during the period where there is 200-to 300-fold increase in the level of starch.…”

Section: Discussionmentioning

confidence: 99%

“…Developing kernels of normal (W64A inbred), waxy (W64A, BC-8XD), and amylose-extender (W64A, BC-8X2) maize (Zea mays) were harvested from the field 8 Carbohydrate Determinations. To 0.5 g of frozen kernels were added 5 ml of 75% (v/v) ethanol.…”

Section: Methodsmentioning

confidence: 99%

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

et al. 1973

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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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“…ASSAY OF STARCH-BRANCHING ENZYME Branching enzyme activity was determined by the stimulation of a-glucan formation from glucose-l-P by phosphorylase a as already described (6,7). A unit of activity is defined as in HCI with reagent H-Cl and hydrolyzed for 1 h at 100 C. The samples were then frozen and lyophilized.…”

Section: Materlusmentioning

confidence: 99%

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

Boyer

Preiss²

1979

Plant Physiol.

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Chromtography of extracts of maize on diethylaminoethyl-celllose resolvestarc synthae avity into two frcio (Ozbum, Hawker, Preiss 1971 Plant Physlo 4& 785-769). Only starc synthase I is capble of synthesis in the absece of added primer and the presene of 0.5 molar citrate. is enzym fratio has been purified about 1,000fold frm maize kernels for the eosrmnuitant amykAw-exseder (ae). Previous reports have shown glycogen and starch synthase fractions from a number of sources catalyze a-glucan synthesis in the absence of added primer, when in the presence of high concentrations of citrate (4,6,8). This reaction has been termed "unprimed" starch and glycogen synthesis. Citrate (0.5 M) was found to lower the Km ofthe unprimed spinach leafstarch synthase 3,400-fold for glycogen and 270-fold for amylopectin (6 Two fractions of starch synthase have been isolated from maize endosperm by DEAE-cellulose chromatography (10). Starch synthase I is able to catalyze unprimed glucan synthesis in the presence of high citrate concentration whereas starch synthase II is inactive under these conditions. In addition, starch synthase I had greater activity with glycogen than with amylopectin as primer, while starch synthase II had greater activity with amylopectin. Schiefer et al. (13) showed that treatment of a starch synthase from sweet corn with a-amylase abolished the unprimed starch synthesis activity and suggested that primer had been removed. However, they were unable to characterize fully the high citrate reaction, because their preparations were contaminated with branching enzyme. The purpose of this report is to describe the properties of the citrate-stimulated starch synthase reaction of maize endosperm. The purification of starch synthase I virtually free of branching enzyme activity was made possible by using endosperm extracts of the branching enzyme mutant amyloseextender. A preliminaqr report of some of this work has already appeared (I 1). MATERIALS AND METHODS MATERLUSThe dent maize (Zea mays L.) inbred W64A and inbred W64A homozygous for the endosperm mutant amylose-extender (ae) were field grown in 1976. Plants were self-or sib-pollinated and ears were harvested at 22 days after pollination, quick frozen in dry ice, and stored at -15 C until used. ADP-['4Cjglucose was prepared enzymically (12). Maize amylopectin (amylose-free) was purchased from Calbiochem; glycogen, ,B-amylase, and rabbit muscle phosphorylase a were obtained from Sigma. AminoalkylSepharose resin was prepared by the procedures of . All other reagents were of the highest purity available.PURICATION OF ADP-GLUCOSE -STARCH SYNTHASE Kernels (230 g) were ground with a porcelain mortar and pestle in the presence of 100 ml of cold 50 mm Tris-acetate buffer (pH 7.5) containing 2.5 mm DTT and 10 mm EDTA. Incompletely ground endosperm and pericarp material was collected by passing the homogenate through five layers of cheesecloth and the grinding repeated two additional times. This extracted solution constituted the crude extract. AM additional proce...

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The De Novo Synthesis of Polysaccharide by Phosphorylase

Cited by 73 publications

References 2 publications

Phosphorylases I and II of Maize Endosperm

Phosphorylases I and II of Maize Endosperm

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

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