Characterization of Pea Chloroplast D-Enzyme (4-α-d-Glucanotransferase)

Kakefuda, Genichi; Duke, Stanley H.

doi:10.1104/pp.91.1.136

Cited by 45 publications

(42 citation statements)

References 27 publications

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“…1). Although we found 1-enzyme to be relatively high in activity in pea petals (603 nmol min-' g-' fresh weight) and it is in high activity in leaf tissues (15,16), it probably would not contribute much to total amylolytic activity, except when P04-was present for starch phosphorylase activity (15). However, even if starch phosphorylase had been included in the total amylolytic activity measurement of pea tissues it would not greatly contribute to the total amylolytic activity.…”

Section: Quantitation Of Hydrolytic Starch Degrading Enzymes In Leavecontrasting

confidence: 70%

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Saeed

Duke²

1990

Plant Physiol.

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Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or ,B-limit dextrin containing gel and KI/12 staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar fI-amylase (EC 3.2.1.2) and the primarily apoplastic aamylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily Bl-amylase) activity and considerably higher ratios of ,-to aamylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and fl-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of a-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher a-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in aamylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the a-amylase that was greatly increased in activity was the primarily apoplastic aamylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic a-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar 8-amylase activity.The physiological roles of amylases in vegetative tissues of higher plants are poorly understood. One reason for this current state of confusion is that at the cellular level most of the amylolytic activity in plant vegetative tissues is extrachloroplastic, away from the site of starch synthesis and degradation. More than 90% of the total amylolytic activity in pea leaves has been reported to be extrachloroplastic (16).

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Section: Quantitation Of Hydrolytic Starch Degrading Enzymes In Leavecontrasting

confidence: 70%

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Saeed

Duke²

1990

Plant Physiol.

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“…Although the conditions existing within cotyledons ofgerminating peas (e.g. pH, starch concentration, compartmentation of a-amylase, and starch) remain undefined, under our assay conditions, which appeared to be optimal, 12 …”

Section: End Product Analysismentioning

confidence: 87%

“…Samples were then centrifuged for 5 min at maximum rpm in a microcentrifuge (Beckman, microfuge 11). Sugars, maltodextrins and oligosaccharides in 40 ,gL samples were separated by HPLC and detected as before (12), except that the column flow rate was 0.2 mL/min. Analysis of hydrolysis products of maltodextrins (maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) (Boehringer Mannheim) by a-amylase was conducted in the same manner, except that the concentration of substrates was 2 mg/mL.…”

Section: End Product Analysesmentioning

confidence: 99%

Characterization of α-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

Beers

Duke²

1990

Plant Physiol.

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The most abundant a-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This a-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an a-amylase by polarimetry, substrate specificity, and end product analyses. The purified aamylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This a-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/ chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein.Common cell wall constituents (e.g. fi-glucan) are not substrates of the enzyme. A very low amount of this a-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or a-amylase which is integrally associated with the chloroplast.

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“…In the cytosol, maltose metabolism requires a 4-␣-glucanotransferase (EC 2.4.1.25). This enzyme was called the cytosolic D enzyme and, by analogy with the plastidial D enzyme, was believed to have no activity with maltose (12). Arabidopsis thaliana plants lacking this enzyme accumulate up to 100-fold more maltose than wild type and grow significantly more slowly (13,14).…”

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confidence: 99%

Domain Characterization of a 4-α-Glucanotransferase Essential for Maltose Metabolism in Photosynthetic Leaves

Steichen

Petty

Sharkey

2008

Journal of Biological Chemistry

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Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-␣-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of ϳ150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the ␤-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.During photosynthesis, as much as one-half of the carbon fixed during the day is stored as transitory starch inside chloroplasts for remobilization at night. The pathway by which starch in leaves is converted to sucrose has only recently been elucidated (1) and some questions remain. The breakdown of storage starch upon seed germination takes place essentially outside of cells (cellular integrity is lost) while transitory starch must be broken down in intact chloroplasts and cells (2). Transitory starch must have some phosphate attached in order to be remobilized at night (3-6). During the day, carbon is exported from the chloroplast almost exclusively as triose phosphate, but at night maltose is exported (7,8). Starch is acted on by ␤-amylase (9), and the resulting ␤-maltose is exported from chloroplasts through a novel maltose transporter (10). Plants lacking this transporter accumulate maltose in their plastids (11). In the cytosol, maltose metabolism requires a 4-␣-glucanotransferase (EC 2.4.1.25). This enzyme was called the cytosolic D enzyme and, by analogy with the plastidial D enzyme, was believed to have no activity with maltose (12). Arabidopsis thaliana plants lacking this enzyme accumulate up to 100-fold more maltose than wild type and grow significantly more slowly (13,14). Potato plants in which this enzyme was reduced in activity accumulated high levels of maltose in leaves but leaf starch synthesis and starch metabolism in tubers was not affected (15).This enzyme is now called disproportionating enzyme 2 (DPE2) 3 because of its similarity to the disproportionat...

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Characterization of Pea Chloroplast D-Enzyme (4-α-d-Glucanotransferase)

Cited by 45 publications

References 27 publications

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Characterization of α-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

Domain Characterization of a 4-α-Glucanotransferase Essential for Maltose Metabolism in Photosynthetic Leaves

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