Acid Polyacrylamide Gel Electrophoresis Research Articles

Allelic variation at the storage protein loci of 55 US‐grown white wheats

AbstractFifty soft white and hard white wheat cultivars (Triticum aestivum L.), and five club wheat cultivars (T. compactum L.) were partially characterized in terms of their storage protein compositions, i.e. gliadins, and high molecular weight and low molecular weight glutenin subunits (HMW‐GS and LMW‐GS, respectively). At the Glu‐1 loci, HMW‐GS composition 1,7 + 9,2+ 12 was found to be predominant, being expressed in 11 cultivars out of 55. The most common alleles at the loci coding for gliadins and LMW‐GS were found to be Gli‐A1/Glu‐A3a (43.6%), Gli‐B1/Glu‐B3b (36.4%), Gli‐D1a/Glu‐D3a (38.1%) and Gli‐Dli/Glu‐D3a (21.8%). Two‐dimensional fractionation (acid‐poly‐acrylamide gel electrophoresis (A‐PAGE) × sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE)) of reduced and alkylated glutenins revealed that the number and the relative mobility of LMW‐GS polypeptides were different from those reported for the corresponding Glu‐3 alleles of hard‐bread wheat cultivars. This result could account for the different technological properties of soft white wheats compared with hard‐bread wheat cultivars, owing to the major impact of LMW‐GS on dough quality.

Immunoblotting of gliadins separated by acid PAGE: Analysis of electrotransference conditions

Several transference buffers and conditions (time and voltage) were tested to establish a suitable procedure for immunoblotting of gliadins after acid polyacrylamide gel electrophoresis (A‐PAGE). Ethanolic extracts of wheat flour were separated by A‐PAGE, transferred to nitrocellulose membranes and detected using an anti‐gliadin serum. The blot resolution was highly dependent on the buffer employed for electrotransference. The best results were obtained when a 10 mM‐H3PO4/NaH2PO4 buffer, pH 2.7, was employed. All gliadin fractions were transferred using this buffer. Sharp bands were obtained and good resolution was achieved.

Possible role of isoaspartyl methyltransferase towards regulation of acid trehalase activity in Saccharomyces cerevisiae

Logarithmically growing cells of S. cerevisiae contained high neutral trehalase (NT) activity while stationary-phase cells had high acid trehalase (AT) activity. Change in activity profile of AT and NT were different during growth under different conditions, particularly during growth in acetate medium and up to 1 h of germination period, but that for AT and isoaspartyl methyltransferase (IMT) were found to be almost identical. Concomitant increase in NT activity as well as increase in cAMP level was noticed at the onset of spore germination. Increase in AT and IMT activities as well as decrease in S-adenosyl- l-methionine (AdoMet) level were noticed during stationary phase of growth. Acidic polyacrylamide gel electrophoresis and subsequent autoradiography revealed that substrate of IMT was a protein of molar mass around 82 kDa which could be an AT. Methylated AT was found to be more active while non-methylated AT was relatively less active in comparison to the untreated sample. Since AT existed as an equilibrium mixture of protomer and oligomer, it was suggested that IMT catalysed carboxyl methylation might have some contribution towards the regulation of AT activity.

The use of acid Polyacrylamide gel electrophoresis to determine the effect of locality of growth on the protein profiles of barley, wheat and honeybush tea

Acid Polyacrylamide gel electrophoresis (A-PAGE) of proteins was used to identify different spring wheat cultivare, barley cultivars and Cyclopia species. Experimental conditions for the extraction of the proteins from single kernels as well as corresponding flour samples are described. Different solvents for extracting the proteins are mentioned. An electrophoretic formula was prepared for each cultivar and species using relative band mobility and staining intensities of protein bands. Distinctive band patterns were obtained. Cultivars and species collected on different locations were subsequently analysed.

Plasma protease inhibitor (PI) system in the laboratory opossum, Monodelphis domestica.

Protease inhibitor (PI) polymorphism was observed in the laboratory opossum, Monodelphis domestica, by either one-dimensional acid polyacrylamide gel electrophoresis (PAGE; pH 4.6) or isoelectric focusing (pH 3.5-5.0) followed by immunoblotting with rabbit antiserum to human alpha 1-antitrypsin; but acid PAGE produced superior resolution of the PI proteins. Family studies demonstrated an inheritance of nine codominant autosomal alleles, PID, PIE, PIF, PIG, PIH, PII, PIJ, PIK, and PIM, and a population study revealed frequencies of 0.411, 0.010, 0.341, 0.034, 0.023, 0.071, 0.035, 0.020, and 0.055, respectively.

Linkage mapping of ‘25-kDa globulin’ genes on homoeologous group-1 chromosomes of bread and durum wheat

Acid polyacrylamide-gel electrophoresis (A-PAGE) of ethanol-soluble proteins from the endosperm of bread and durum wheats reveals some bands encoded by genes on the homoeologous group-1 chromosomes with higher mobility than the α-gliadins. The isolation of these proteins showed that they were the previously described '25-kDa globulins' encoded by genes at the Glo-A1, Glo-B1, and Glo-D1 loci. The variability found among a collection of 51 bread and 81 durum wheats was very low: two allelic variants at Glo-A1 and no variants at Glo-B1 in each of the two species, and two allelic variants at Glo-D1 in bread wheat. Inheritance studies of '25-kDa globulin' genes on group-1 chromosomes of bread and durum wheat were carried out on the F2 progeny from four crosses, two in bread wheat and two in durum wheat. The linkage mapping of the 1A '25-kDa globulin' genes of bread wheat was done based on four prolamin loci: Glu-A1, Glu-A3, Gli-A1 and Gli -A3. The percentages of recombination and the distances found allowed a re-evaluation of the linkage map of endosperm protein loci on this chromosome. The Glo-A1 locus was found to be located at the distal end of the short arm of 1A chromosome, at a distance of 5.23±1.99 cM from Gli-A1, 6.85±2.22 cM from Glu-A3, 22.64±3.62 cM from Gli-A1, and at a recombination percentage of 49.30±4.40 from Glu-A1. A similar distance between Gli-A1 and Glo-A1 (4.82±1.75 and 6.66±2.26 cM) was found in durum wheat. The distance between Gli-D1 and Glo-D1 on chromosome 1D was 2.86±1.39 cM.

Electrophoretic Characterization of Spring Spelt Wheat Gliadins

Gliadin compositions of 95 spring spelts, 5 winter spelts, and Neepawa Hard Red Spring (HRS) wheat were evaluated by acid-polyacrylamide gel electrophoresis (A-PAGE). Five spring spelts grown in three environments over 2 years were compared to the HRS cultivar Katepwa using A-PAGE and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the scoring of 13 bands, the spring and winter spelts diverged widely from Neepawa HRS wheat. None of the spring spelts evaluated had a gliadin protein profile identical to that of commercially available winter spelt. Spring and winter spelts were differentiated from Katepwa HRS wheat based on ω-gliadin composition, in which common wheat was characterized by the presence of a slow-moving ω-gliadin and a strong-staining fast-moving ω-gliadin. Common wheat and spring spelt accessions SK0505, SK0263, and RL5407 (black-hulled spelts) had a distinct strong band in the γ-gliadin region with MW of about 64K which was absent in winter spelts and the spring spelts SK0021 and PGR8801 (white-hulled spelts). The molecular weights of gliadin proteins were in the range of 34K-75K.

Identification of glycosylated forms of wheat storage proteins using two-dimensional electrophoresis and blotting.

Two-dimensional electrophoresis with acid-polyacrylamide gel electrophoresis (PAGE), followed by sodium dodecyl sulfate (SDS)-PAGE and SDS-PAGE of unreduced polypeptides followed by SDS-PAGE under reducing conditions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncovalent polymers of glutenins. Gels were blotted under semidry conditions according to Laurière (Anal. Biochem. 1993, 212, 206-211) to allow large polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbeck and Hösel (Glycoconjugate J. 1990, 7, 63-74), or using anti-(xylose-containing N-glycan) antibodies (Laurière et al., Plant Physiol 1989, 90, 1182-1188). High and low molecular weight glutenin subunits were shown to aggregate through both disulfide bridges and noncovalent protein-to-protein interactions. Aggregated gamma-gliadins were also demonstrated. Glycans were detected on both gliadin and glutenin polypeptides. Covalently aggregated low molecular weight glutenins were shown to contain N-glycans with xylose, which demonstrated their sorting in the Golgi apparatus.

Toxicity of Bread Wheat Lines Lacking Prolamins Encoded by theGli-B1/Gli-B5/Glu-B3andGli-D1/Glu-D3Loci in Coeliac Disease as Determined by their Agglutinating Activity

Peptic-tryptic (PT) digests of alcohol-soluble proteins from the flour of three mutant lines of bread wheat, lacking γ-gliadins, γ-gliadins and low molecular-weight glutenin subunits encoded by the Gli-B1/Gli-B5/Glu-B3 loci (line S. Pastore 4A), the Gli-D1/Glu-D3 loci (line Alpe 1I-) or both groups of loci (line DM 22166), were compared with those of the normal cultivars S. Pastore and Alpe 1 I for their agglutinating activities on human myelogenous leukemia K562(S) cells, agglutination being strongly associated with toxicity for the coeliac intestine. All of the genotypes tested contained A-type α-gliadins, which constituted about 19% of the gliadins in the S. Pastore and Alpe 1I cultivars, 24.5% in the S. Pastore 4A and Alpe 1I null lines, and 34.8% in the double mutant line, DM 22166, as determined by densitometric scanning of their acid polyacrylamide gel electrophoresis patterns. The minimal concentrations of PT digest required to agglutinate 100% of K562(S) cells were 73mg/l and 96mg/l, in the S. Pastore and Alpe 1I cultivars, respectively, compared with 146mg/l, 138mg/l and 200mg/l in the “null” lines, S. Pastore 4A, Alpe 1 I-and DM 22166, respectively. The results indicated that proteins other than α-gliadins are involved in the gluten-sensitive enteropathy.

Genetic distance between Triticum timopheevi Zhuk., T. aramticum Jakubz. and T. aestivum L.

AbstractThe genetic distances between two cultivated wheat (Triticum aestivum L.) varieties (Martonvásári 9, Martonvásári 15), a Martonvásári 9 line possessing the crossability gene krl, and 21 accessions of T. timopheevi Zhuk. and T. araraticum Jakubz. were estimated, based on agro‐morphological, physiological and biochemical analysis of data. Cluster analysis based on Mahalanobis' D2 values was applied. All 21 accessions of T. timopheevi and T. araraticum could be classified into eight clusters. Clusters I and II consisted of all the T. timopheevi, while T. araraticum was located in six clusters. Discriminant analysis was applied to test significant differences between cluster pairs. The genetic distance (GD) based on the electrophoretic data of gliadins indicated two types of electrophoregrams in T. timopheevvi, distinguished as groups A and B. T. araraticum accessions were variable as regards the spectra. Mean, minimum and maximum GD were estimated within and between different wheat groups based on acid polyacrylamide gel electrophoresis.

Placement of loci for avenins and resistance to Puccinia coronata to a common linkage group in Avena strigosa

Alcohol-soluble seed storage proteins of oat (avenins) were extracted from two diploid accessions representing the A genome and separated by high-resolution acid polyacrylamide gel electrophoresis. Polymorphisms were detected for three clearly resolved protein bands. Linkage analysis of 88 F2:3 families mapped the three bands to a single locus. Integration of avenin segregation data with an RFLP linkage map constructed from the same population, mapped the avenin locus to a linkage group containing a locus conferring resistance to nine isolates of Puccinia coronata. Linkage between genes encoding alcohol-soluble seed proteins and genes for resistance to Puccinia species was also observed for the homoeologous group 1 chromosomes of barley (1H), rye (1R), wheat (1A, 1B, 1D), and chromosomes 4 and 10 of maize.

A Null Gli-D1 Allele with a Positive Effect on Bread Wheat Quality

Abstract The French wheat cultivar Darius (Da) has very good bread-making quality, even though it possesses the high M r glutenin subunit combination 2, 7 and 12, which is associated with poor quality, and a null allele at the Gli-D1 locus. Darius was crossed with three cultivars, Corin (Cor), Capitole (Cap) and Courtot (Cou), of poor, medium, and good quality, respectively. The three progenies (Cor × Da, Cap × Da and Da × Cou) were used to investigate the genetic basis of the good quality of Darius. Gliadin and glutenin compositions were analysed by acid polyacrylamide gel electrophoresis (A-PAGE) and SDS-PAGE, respectively, from half F2 grains, and the quality was evaluated using six technological criteria for the corresponding plants. The high M r glutenin subunit alleles of Darius produced a negative effect on quality. The null allele of Darius, characterised by the absence of the Gli-D1 encoded ω-gliadins, was associated significantly with higher dough tenacity P , and strength W (up to 40% in the Cor × Da progeny). Darius had a higher amount of B zone low M r glutenin subunits than the three other cultivars. The null allele of Darius reduced the dough extensibility in the two first progenies, and probably increased the ratio of aggregated glutenin to unaggregated gliadins. These results demonstrated that using only one locus breeders can improve particular quality traits.

Erratum: Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation

Erratum: Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation

Differences in accumulation of storage proteins between wheat cultivars during development

AbstractThe concentration and composition of storage proteins in the developing kernels of five different wheat cultivars were investigated. The plants were grown in solution culture under strictly controlled conditions in climatic chambers and with long‐term limitation of external nitrogen availability. Thus, by growing these cultivars under the same defined treatments, reliable differences in the storage protein development could be determined. The total protein concentration was assayed using an automatic nitrogen analyser. The storage protein composition was examined by acidic polyacrylamide gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis from 6 to 58 days after anthesis. The results showed that, in all stages of plant development, the ranking of the cultivars as to the storage protein concentration was the same as for mature field‐grown kernels. The differences in the storage protein concentration between the cultivars could not be explained by differences in protein composition. The start of storage protein formation during plant development did not differ between the tested cultivars, as determined by the electrophoresis pattern. The differences in the storage protein concentration between cultivars most likely depends on the relative balance between starch and nitrogen deposition in the grain, during the whole grain filling period.

Erratum

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Toyopearl ion-exchange chromatography. Constituent globin chains, α1, α2, β1 and β2, were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reversed-phase column. Tryptic peptide mapping indicated that the α-globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the β1-globin chain has a primary structure different from that of the β2-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (α1 α2 β 2 1 ), CII (α1 α2 β1 β2), and CIII (α1 α2 β 2 2 ). Hemoglobin CII was a hybrid between the two types each of α- and β-chain and could be constructed in vitro from two hemoglobin components CI and CIII.

Subunit structure of multiple hemoglobins in carp.

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Tokyo-pearl ion-exchange chromatography. Constituent globin chains, alpha 1, alpha 2, beta 1, and beta 2, were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reverse-phase column. Tryptic peptide mapping indicated that the alpha-globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the beta 1-globin chain has a primary structure different from that of the beta 2-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (alpha 1 alpha 2 beta 1(2)), CII (alpha 1 alpha 2 beta 1 beta 2), and CIII (alpha 1 alpha 2 beta 2(2)). Hemoglobin CII was a hybrid between the two types each of alpha- and beta-chain and could be constructed in vitro from two hemoglobin components CI and CIII.

Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation

Gliadin allele compositions of 21 Canadian spring common wheat cultivars, most of which belong to the Canada western red spring (CWRS) class, were studied and great similarity in their genotypes was confirmed. It was found that alleles frequent in the set of Canadian wheats (such as Gli-B1d, Gli-D1j, Gli-A2m, and Gli-D2h) are very rare or absent in common wheat cultivars from other regions and countries studied earlier, indicating that germplasm of CWRS cultivars is rather unique. It may be suggested that alleles frequent in Canadian cultivars relate to important technological characteristics of these wheats and may possibly serve as marker genes during selection for quality traits. Similarity of gliadin electrophoregrams obtained by two different acid polyacryl-amide gel electrophoretic procedures for the same genotype was established, and the component composition of allelic variants of blocks of gliadin components found in the set of Canadian cultivars and in standard cultivars Chinese Spring and Bezostaya 1 are described.

Regulation of neutrophil elastase activity by elastin-derived peptide

To understand the interaction between elastin and elastase, elastin from human aorta was incubated with human leukocyte elastase under conditions favoring proteolysis. Low molecular weight species were separated from the protein fraction by a small centrifuged gel filtration column. The only product of the elastin digest detected on acid polyacrylamide gel electrophoresis was a single band of slower cathodal mobility than human leukocyte elastase alone. This band cross-reacts with antibody to human elastase, indicating that the slow migrating band contains elastase. The putative human leukocyte elastase-elastin-derived peptide complex was treated with hydroxylamine to cleave any possible acyl-enzyme complexes and was then measured for amidolytic activity. Analysis of the amino acid composition of elastin-derived peptide indicates the presence of alanine, glycine, and richness in hydrophobic residues, suggesting that these residues are involved in elastase interaction(s). Incubation of the elastase-elastin-derived peptide with alpha 1-protease inhibitor causes dissociation of the complex and formation of an elastase-alpha 1-protease inhibitor complex. Our results suggest that, locally at the site of proteolysis, elastase activity may be regulated by elastin-derived peptide(s) during elastinolysis.

Comparison of High-molecular-weight Subunits of Glutenin and Baking Performance of Flours Varying in Bread-making Quality

Baking experiments and analyses of gluten protein composition were carried out with flours from U.S. and Norwegian wheat lines differing in baking characteristics. Bread-making quality was evaluated by mixograph and baking tests. The compositions of high-molecular-weight (HMW) glutenin subunits and gliadins were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and acid-polyacrylamide gel electrophoresis, respectively. Lack of correlation between the Glu-1 quality score based on HMW glutenin subunits and backing performance was observed. For the Norwegian wheat lines, variation in gluten polypeptides encoded from the short arm of the 1B chromosome partially explained variation in baking performance.

Heterogeneity in porcine pituitary luteinizing hormone: amino acid and carbohydrate analysis.

Pituitary LH from porcine pituitary glands was purified by a buffered ethanol extraction procedure, ion exchange on DEAE- and carboxymethyl-cellulose, and molecular exclusion on Sephacryl S-200. Purity was assessed by amino acid composition, N-terminal sequence, and polyacrylamide gel electrophoresis. Subunits were isolated by countercurrent distribution and reverse phase HPLC. Four major forms of the alpha-subunit were detected: 1-96 (50%), 3-96 (23%), 4-96 (16%), and 7-96 (11%). [The original sequence report described only the 7-96 form, but we have detected the other forms in our studies of porcine FSH and in this and other species of LH.] Comparable N-terminal heterogeneity was not observed for the beta-subunit. Additional heterogeneity was observed for both subunits, attributable to heterogeneity in the N-linked oligosaccharide moieties. The isolated subunits were submitted to detailed compositional carbohydrate analysis, using pulsed amperometric detection of the HPLC-resolved sugar monomers after trifluoroacetic acid hydrolysis. Sialic acid and sulfate esters were estimated on separate hydrolyzates. The compositional data suggest that the two alpha-subunit N-linked moieties are hybrid complex biantennary structures with sulfated N-acetylgalactosamine (40-50%). Sixty to 70% of the alpha-subunit oligosaccharides are fucosylated. The beta-subunit of porcine LH has a single glycosylation site, which contains a mixture of biantennary oligosaccharide chains (80-90%) ending in N-acetylgalactosamine, half of which are sulfated. The balance (10-20%) are hybrid chains ending in sialylated galactose. The majority of the oligosaccharide on the beta-subunit is fucosylated.