Resolution of High Molecular Weight Glutenin Subunits by a New SDS‐PAGE System Incorporating a Neutral pH Buffer

Kasarda, Donald D.; Woodard, Kelly M.; Adalsteins, A. Elva

doi:10.1094/cchem.1998.75.1.70

Cited by 43 publications

(33 citation statements)

References 4 publications

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“…A protein band appears at 53 kDa in both spring emmer and autumn emmer, in contrast to common wheat. This protein band was previously observed in spelt wheat cultivars by Dvořáček and Čurn (2003) and could be attributable to ω-gliadins which have a molecular weight ranging from 44 kDa to 74 kDa (Kasarda et al 1998). ω-Gliadins were classified by Shewry et al (1986) as compounds poor in sulphur content with scarce tendency to form disulphide bounds.…”

Section: Quality and Composition Of Grains And Floursmentioning

confidence: 69%

Composition, protein contents, and microstructural characterisation of grains and flours of emmer wheats (Triticum turgidum ssp. dicoccum) of the central Italy type

Giacintucci¹,

Guardeño²,

Puig³

et al. 2014

Czech J. Food Sci.

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Giacintucci V., Guardeño L., Puig A., Hernando I., Sacchetti G., Pittia P. (2014) The microstructural characteristics were evaluated of two types of Italian Farro (Triticum turgidum ssp. dicoccum) with spring and autumn growth habits, the former with a vitreous tendency and the latter with a floury tendency. Common wheat flours and grains (Triticum aestivum) were used as controls. Protein fractions such as glutenin and gliadin were extracted from Triticum turgidum ssp. dicoccum flours and studied by SDS-PAGE in order to make a comparison between the electrophoretic analyses and microstructural studies which were conducted on the same samples using Scanning Electron Microscopy (SEM and Cryo-SEM). The results obtained by SDS-PAGE showed that the gliadin patterns of both emmer samples were similar, while the common wheat gliadins showed a band at 90 kDa that was not present in the gliadin fraction of emmer. When the glutenin patterns were analysed, the autumn emmer did not show the low molecular weight protein bands (16-23 kDa) whilst spring emmer wheat appeared more similar to common wheat. Regarding the microstructural characteristics of the kernels, spring (vitreous tendency) emmer showed starch granules covered by protein to a higher extent than autumn emmer. These differences were also observed in flours. The gluten of spring emmer wheat was observed as a homogeneous structure showing similarities with common wheat gluten, while autumnal emmer gluten appeared more heterogeneous and lacking in structure.

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Section: Quality and Composition Of Grains And Floursmentioning

confidence: 69%

Composition, protein contents, and microstructural characterisation of grains and flours of emmer wheats (Triticum turgidum ssp. dicoccum) of the central Italy type

Giacintucci¹,

Guardeño²,

Puig³

et al. 2014

Czech J. Food Sci.

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“…An important improvement was the use of neutral pH conditions (pH 7.0) during electrophoresis, instead of the alkaline pH (pH 8.2e8.5) of the resolving gel from the Tris-glycine system, leading to an improved protein stability (Moos et al, 1988). As such, a discontinuous Bis-Tris electrophoresis system with a neutral operating pH led to an excellent resolution after electrophoretic separation of the HMW-GS under investigation (Kasarda et al, 1998). Furthermore, based on the band intensities in these Bis-Tris gels, it was observed that subunits Ax1 and Dx5 and Ax2* and Dx5, respectively, switched position in comparison to their positions in the Trisglycine system at pH ca.…”

Section: Introductionmentioning

confidence: 90%

“…SDS-PAGE was carried out according to Kasarda et al (1998) with some modifications including a homogeneous NuPAGE 10% polyacrylamide e Bis-Tris [bis (2-hydroxyethyl) iminotris(hydroxymethyl) methane-HCl] gel at pH 6.4, 1.0 mm Â 10 well (Invitrogen, Carlsbad, CA, USA) and a MOPS-Tris (50 mmol/L MOPS, 50 mmol/L Tris, 3.5 mmol/L SDS, 1 mmol/L EDTA, pH 7.7) running buffer containing DTT (5 mmol/L) as reducing agent added to the inside chamber. Flour (20 mg) was extracted with 1 mL of extraction buffer (293.3 mmol/L sucrose, 246.4 mmol/L Tris, 69.4 mmol/L SDS, 0.51 mmol/L EDTA, 0.22 mmol/L serva blue G250, 0.177 mmol/L phenol red, 0.105 mmol/L HCl, pH 8.5) for 24 h under reducing conditions (DTT, 50 mmol/L).…”

Section: Sds -Polyacrylamide Gel Electrophoresis (Sds-page)mentioning

confidence: 99%

A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

Lagrain

Rombouts

Wieser

et al. 2012

Journal of Cereal Science

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a b s t r a c tThe high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 z 3 > 1 > 6 z 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/ MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SDS-PAGE.The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order.

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“…The second dimension was developed with a NuPage 4 -12% Bis-Tris Zoom gel. Gels were stained with Coomassie brilliant blue G-250 (63).…”

Section: D Gel Electrophoresismentioning

confidence: 99%

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

Balmer

Vensel

Cai

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

161

137

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A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxin͞Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxinTrx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.redox regulation ͉ target proteins ͉ ferredoxin-thioredoxin reductase O ur understanding of the function of the regulatory disulfide protein thioredoxin (Trx) has increased dramatically in the past few years, with the advent of new methodologies. Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2-4). These capabilities have defined an extended role of Trx in chloroplasts (1, 5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4, 9). Currently almost 200 proteins appear to be linked to Trx in plants (10). One major organelle that has been neglected, however, is the amyloplast, a plastid of heterotrophic tissues that performs a wide range of biosynthetic reactions, including the synthesis and storage of abundant quantities of starch.To help fill this gap, we have applied proteomic and immunological approaches to investigate the occurrence and function of Trx in amyloplasts. We now report evidence that amyloplasts isolated from wheat starchy endosperm resemble chloroplasts in containing a complete ferredoxin͞Trx system composed of ferredoxin, ferredoxin-Trx reductase (FTR), and Trx (m-type). Application of affinity chromatography and fl...

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Resolution of High Molecular Weight Glutenin Subunits by a New SDS‐PAGE System Incorporating a Neutral pH Buffer

Cited by 43 publications

References 4 publications

Composition, protein contents, and microstructural characterisation of grains and flours of emmer wheats (Triticum turgidum ssp. dicoccum) of the central Italy type

Composition, protein contents, and microstructural characterisation of grains and flours of emmer wheats (Triticum turgidum ssp. dicoccum) of the central Italy type

A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

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