Nanomechanical force measurements of gliadin protein interactions.

The strength and nature of interactions between monomeric gliadin proteins involving alpha-alpha, omega-omega, and alpha-omega interactions in 0.01M acetic acid, and the effect of urea has been investigated. It was shown by means of nanomechanical force measurements that the stretching events in the separation curve after adhesive phenomena originated from proteins.

Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes.

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T3, T4, T5) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and "added" subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots.

Characterization of the emulsification properties of 2S albumins from sunflower seed.

The ability of 2S albumins from sunflower seeds to stabilize oil-in-water emulsions has been investigated, demonstrating that one of the proteins (SFA8) effectively stabilizes emulsions, while another (SF-LTP) does not stabilize emulsions. The surface tension and surface dilation viscosity of these two proteins were measured, rationalizing the emulsifying ability of SFA8 in terms of its ability to form a strongly elastic monolayer at interfaces. The secondary structure changes that occur upon adsorption of SFA8 to the oil/water interface have also been studied by fluorescence, circular dichroism (CD), and Fourier-transform infrared (FT-IR) spectroscopy.

The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris.

Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity. We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds. Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E.

Prolamin aggregation, gluten viscoelasticity, and mixing properties of transgenic wheat lines expressing 1Ax and 1Dx high molecular weight glutenin subunit transgenes.

The composition of high molecular weight (HMW) subunits of glutenin determines the gluten strength and influences the baking quality of bread wheat. Here, the effect of transgenes coding for subunits 1Ax1 and 1Dx5 was studied in two near-isogenic wheat lines differing in their HMW subunit compositions and mixing properties. The subunits encoded by the transgenes were overexpressed in the transformed lines and accounted for 50-70% of HMW subunits.

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli.

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography.

Extraction, separation, and purification of wheat gluten proteins and related proteins of barley, rye, and oats.

The wheat proteins, which are active in celiac disease and other glutenrelated conditions, are defined as prolamins, in that they are soluble as individual subunits in alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60-70% (v/v) aqueous ethanol. However, in wheat grain and flour, about half of these subunits are present in polymers that are not soluble in alcohol-water mixtures unless disulfide bonds between the component subunits are reduced using an agent such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). These alcohol-insoluble polymers are traditionally called glutenins and the related alcohol-soluble monomers are called gliadins; the two groups of proteins together form the major part of the gluten fraction.

Studies in vivo of omega-gliadins in gluten sensitivity (coeliac sprue disease).

1. Highly purified omega-gliadins from wheat were used to challenge gluten-sensitized individuals. Characteristic responses by mucosal CD3(+) and gamma delta+ lymphocytes were demonstrated.

Identification of the major water/salt insoluble wheat proteins involved in cereal hypersensitivity.

Background: Several studies have investigated water/salt soluble proteins which comprise 50% of the proteins in wheat. The remaining 50% of wheat proteins, are water/salt insoluble proteins of which there is limited information on their role in cereal hypersensitivity.

Objectives: To investigate the allergenicity of the water/salt insoluble gliadin and glutenin proteins (prolamins).

Transformation of wheat with high molecular weight subunit genes results in improved functional properties.

The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes. We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour.

Disulphide structure of a sunflower seed albumin: conserved and variant disulphide bonds in the cereal prolamin superfamily.

Disulphide mapping of a methionine-rich 2S albumin from sunflower seeds showed four intra-chain disulphide bonds which are homologous with those in a related heterodimeric albumin from lupin seeds (conglutin delta). Similar conserved disulphide bonds are also present in alpha-gliadin and gamma-gliadin storage proteins of wheat, but a lower level of conservation is present in a further related group of proteins, the cereal inhibitors of alpha-amylase and trypsin. These differences may relate to the different functions of the proteins.

Immunochemical identification of LMW subunits of glutenin associated with bread-making quality of wheat flours.

A murine monoclonal antibody (IFRN 0067), one of a library developed against prolamin fractions fromTriticum aestivum, has been characterised using a combination of immunoassay and immunoblotting techniques. The antibody was specific for two glutenin polypeptides which appeared by 2-dimensional electrophoresis to belong to the B group of LMW subunits. From results of antibody-binding studies with material extracted from genetic stocks, it was deduced that the target polypeptides were encoded on the short arm of chromosome 1D.

Isolation and partial amino acid sequence of domains of nitrate reductase from spinach.

Fragments of spinach nitrate reductase (NR) were prepared by limited proteolysis of immunopurified enzyme using both Staphylococcus aureus V8 protease and trypsin. Incubation of NR with V8 protease yielded two enzymically active fragments which could be size separated by FPLC on a Superose 12 column or subjected to further proteolysis while bound to a blue Sepharose affinity column. An NADH-ferricyanide (NADH-FR) active fragment bound to, and was eluted from, a blue Sepharose column by micromolar concentrations of NADH.

Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities.

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.

Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase.

The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r.