The hydrolysis of glucose monophosphates by a phosphatase preparation from pea seeds

Turner, Donella H.; Turner, John F.

doi:10.1042/bj0740486

Cited by 41 publications

(14 citation statements)

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“…Since fluoride is an inhibitor of phosphatases (3,16) and has no effect on NR activity (6), the addition of KF to the assay systems should decrease the NADPH-NR but have no effect on NADH-NR. This inhibitory effect of KF was confirmed NADPH pH 6.5 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Specificity for Nicotinamide Adenine Dinucleotide by Nitrate Reductase from Leaves

Wells¹,

Hageman²

1974

Plant Physiol.

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Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPHphosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90 %) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH2PO4 and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).While numerous investigations have shown that nitrate reductase extracted from a variety of higher plants has a specific or highly preferential requirement for NADH (2), the enzyme from soybean leaves apparently utilizes either NADH or NADPH with equal efficiency (1, 6). Investigations with the in vivo assay also suggest that the enzyme is NADH-dependent (12). Although we have reported that NR' from corn was NADH-specific (1, 9), recently we have observed that NR from leaf tissue of certain corn genotypes can also utilize NADPH as the electron donor. The level of NADPH activity observed in these initial experiments was a function of pH and assay buffer.It is possible that the NADPH-NR activity in both soybean (8), and Hevea brasiliensis (10).The objectives were: (a) to demonstrate the range of NADPH-dependent NR activity in relation to NADH-NR activity as affected by the type and pH of the assay buffer; (b) to determine if NR from corn genotypes that exhibited marked NADPH activity is a single enzyme as was shown for the soybean enzyme (1); and (c) to determine if the NADPH-NR activity in crude and partially purified preparations is an artifact arising from the presence of a phosphatase that hydrolyzes NADPH to NADH and P,.MATERIAL AND METHODS Plant Material and Culture. Leaf tissue from corn (Zea miiays L.), soybeans (Glycine max L. Merr. var. Hawkeye), pigweed (Amaranthus hybridus L.), and foxtail (Seteria faberii Herrm.) was harvested from young plants grown in growth chambers (16 hr light-2,500 ft-c at 32 C and 8 hr dark at 22 C) in vermiculite (Zonalite Co., Chicago, Ill.) subirrigated daily with Hoagland's No. 1 nutrient solution. ...

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Section: Resultsmentioning

confidence: 99%

Specificity for Nicotinamide Adenine Dinucleotide by Nitrate Reductase from Leaves

Wells¹,

Hageman²

1974

Plant Physiol.

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“…However, NaF at final concentration of 20 mm was added to inhibit phosphatases (30). The Pi which was formed was determined by the method of Lowry and Lopez (21) with some modifications.…”

Section: Methodsmentioning

confidence: 99%

“…The starch precipitate was suspended in water, filtered through cheesecloth, and diluted to 50 ml with water. A portion of this starch suspension was analyzed for starch content by the anthrone method (30). The rest was used for the preparation of starch granule-bound enzyme according to Leloir et al (20).…”

Section: Methodsmentioning

confidence: 99%

Enzymes of Starch Metabolism in the Developing Rice Grain

et al. 1970

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The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism-a-amylase, 3-amylase, pliosphorylase, Q -enzyme, R -enzyme, and starch synthetase -were assayed in dehulled developing rice grains (Oryza sativa L., variety IR8). Phosphorylase, Q-enzyme, and Renzyme had peak activities 10 days after flowering, whereas a-and ,B-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.Most starch biochemists believe that the starch synthetases (ADP-and UDP-glucose-starch 4-glucosyltransferases) are the enzymes involved in starch synthesis (2,20). Some investigators believe that phosphorylase is also a synthetic enzyme and that the starch synthetase functions to protect the amylose molecule formed with phosphorylase and starch synthetase from being transformed to amylopectin through the action of Q-enzyme (1, 13). Still others propose a multiple pathway synthesis of starch (4).Another unsolved problem in starch biosynthesis is the genetic integrity of amylose in the granule of nonwaxy starches. Previous work on developing rice and corn grains showed that starch synthetase bound to the starch granule occurs mainly in nonwaxy granules (3,20 Q-enzyme (a-1 ,4-glucan:a-1 ,4-glucan 6-glycosyltransferase or branching enzyme), and R-enzyme (amylopectin 6-glucanohydrolase or debranching enzyme)-during grain development in the rice variety IR8. Lines from the same cross differing in amylose content were used to compare enzyme activities in relation to amylose synthesis. The use of such lines grown in the same crop reduces complicating environmental and genetic factors which accompany studies in which different varieties are compared. MATERIALS AND METHODSSamples of developing rice grains (Oryza sativa L., variety IR8) were obtained from the experimental field of the Institute at 7, 10, 14, 21, and 28 days after flowering and immediately stored at 0 C. They were dehulled by hand prior to analysis. The grain weight was determined for each sample.Samples of lines differing in amylose content were grown in a Mylar house in pots containing 6 kg of air-dried soil. One day before transplanting, 10 g of (NH4)2S04 and 8 g Na3PO4 were added to the soil, the soil was mixed well, and the pots were flooded. Four 10-day-old seedlings were transplanted per pot and kept under continuous flooding. Panicles were t...

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“…To measure the total starch content, 50 mg powder was washed three times using 80% ethanol (v/v) and then extracted with 9.2 M and 4.6 M perchloric acid in order. The supernatant was diluted and analyzed by the anthrone method (Turner and Turner, 1960). The apparent amylose content was measured by the iodine colorimetric method as previously described (Juliano, 1971).…”

Section: Starch Content and Chain Length Distribution Analysismentioning

confidence: 99%

The MADS29 Transcription Factor Regulates the Degradation of the Nucellus and the Nucellar Projection during Rice Seed Development

2012

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The MADS box transcription factors are critical regulators of rice (Oryza sativa) reproductive development. Here, we here report the functional characterization of a rice MADS box family member, MADS29, which is preferentially expressed in the nucellus and the nucellar projection. Suppressed expression of MADS29 resulted in abnormal seed development; the seeds were shrunken, displayed a low grain-filling rate and suppressed starch biosynthesis, and contained abnormal starch granules. Detailed analysis indicated that the abnormal seed development is due to defective programmed cell death (PCD) of the nucellus and nucellar projection, which was confirmed by a TUNEL assay and transcriptome analysis. Further studies showed that expression of MADS29 is induced by auxin and MADS29 protein binds directly to the putative promoter regions of genes that encode a Cys protease and nucleotide binding site-Leu-rich repeat proteins, thereby stimulating the PCD. This study identifies MADS29 as a key regulator of early rice seed development by regulating the PCD of maternal tissues. It provides informative clues to elucidate the regulatory mechanism of maternal tissue degradation after fertilization and to facilitate the studies of endosperm development and seed filling.

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The hydrolysis of glucose monophosphates by a phosphatase preparation from pea seeds

Cited by 41 publications

References 13 publications

Specificity for Nicotinamide Adenine Dinucleotide by Nitrate Reductase from Leaves

Specificity for Nicotinamide Adenine Dinucleotide by Nitrate Reductase from Leaves

Enzymes of Starch Metabolism in the Developing Rice Grain

The MADS29 Transcription Factor Regulates the Degradation of the Nucellus and the Nucellar Projection during Rice Seed Development

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