Summary
Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature
Bernardin, J.E. (1978): Gluenin protein interaction with small molecules and ions — the control of flour properties. Bakers Digest 52, 20–23
Bietz, J.A.; Huebner, F.R.; Sandersen, J.E.; Wall, J.S. (1977): Wheat gliadin homology revealed through N-terminal amino acid sequence analysis. Cereal Chem. 54, 1070–1083
Bietz, J.A.; Shepherd, K.W.; Wall, J.S. (1975): Single kernal analysis of glutenin: use in wheat genetics and breeding. Cereal Chem. 52, 513–532
Bietz, J.A.; Wall, J.S. (1972): Wheat gluten subunits: molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. Cereal Chem. 49, 416–430
Bietz, J.A.; Wall, J.S. (1973): Isolation and characterization of gladin-like subunits from glutenin. Cereal Chem. 50, 537–547
Bietz, J.A.; Wall, J.S. (1980): Identity of high molecular weight gliadin and ethanol-soluble glutenin subunits of wheat: relation to gluten structure. Cereal Chem. 57, 415–421
Bloksma, A.H. (1975): Thiol and disulphide groups in dough rheology. Cereal Chem. 52 (3, II), 170r-183r
Charbonnier, L. (1974): Isolation and characterization of ω- gliadin fractions. Biochim. Biophys. Acta 359, 142–151
Crestfield, A.M.; Moore, S.; Stein, W.H. (1963): The preparation and enzymatic hydrolysis of reduced and S-carboxy- methylated proteins. J. Biol. Chem. 238, 622–627
Degani, C.Y.; Patchornik, A. (1971): Selective cyanylation of sulphydryl groups. II on the synthesis of 2-nitro-5-thiocy- anobenzoic acid. J. Org. Chem. 36, 2727–2728
Degani, Y.; Patchornik, A. (1974): Cyanylation of sulphydryl groups by 2-nitro-5-thiocyanobenzoic acid. High yield modification and cleavage of peptides at cysteine residues. Biochemistry 13, 1–11
Duhamel, R.C.; Meezan, E.; Brendel, K. (1980): Metachromatic staining with Coomassie Brilliant Blue R250 of the proline-rich calf thymus histone HI. Biochim. Biophys. Acta 626, 432–442
Edman, P.; Begg, G. (1967): A protein sequenator. Eur. J. Biochem. 1, 80–81
Ewart, J.A.D. (1968): A hypothesis for the structure and rheology of glutenin. J. Sci. Food Agric. 19, 617–623
Ewart, J.A.D. (1972): A modified hypothesis for the structure and rheology of glutenins. J. Sci. Food Agric. 23, 687–699
Ewart, J.A.D. (1977): Re-examination of the linear glutenin hypothesis. J. Sci. Food Agric. 28, 191–199
Ewart, J.A.D. (1978): Glutenin and dough tenacity. J. Sci. Food Agric. 29, 551–556
Field, J.M.; Shewry, P.R.; Miflin, B.J. (1982a): Solubilization and characterization of wheat gluten proteins; correlations between the amount of aggregated proteins and baking quality. J. Sci. Food Agric. (submitted for publication)
Field, J.M.; Shewry, P.R.; Burgess, S.R.; Forde, J.; Parmar, S.; Miflin, B.J. (1982b): The presence of large molecular weight protein aggregates in the protein bodies of developing wheat and other cereal grains. J. Cereal Sci. (subm. for publ.)
Friedman, M.; Krull, L.H.; Cavins, J.F. (1970): The Chromatographic determination of cystine and cysteine residues in proteins as S-β-(4-pyridylethyl)cysteine. J. Biol. Chem. 245, 3868–3871
Hamauzu, Z.; Kamazuka, Y.; Kanazawa, H.; Yonezawa, D. (1975): Molecular weight determination of component polypeptides of glutenin after fractionation by gel filtration. Agric. Biol. Chem. 39, 1527–1531
Hamauzu, Z.; Toyomasu, T.; Yonezawa, D. (1974): Studies on sodium dodecyl sulphate complex of reduced gliadin in relation to the abnormality in SDS-polyacrylamide gel electrophoresis. Agric. Biol. Chem. 39, 1407–10
Holt, L.M.; Astin, R.; Payne, P.I. (1981): Structural and genetical studies of the high-molecular-weight subunits of wheat glutenin. Part 2. Relative isoelectric points determined by two-dimensional fractionation in polyacrylamide gels. Theor. Appl. Genet. 60, 237–243
Huebner, F.R.; Wall, J.S. (1976): Fractionation and quantitative differences of glutenin from wheat varieties varying in baking quality. Cereal Chem. 53, 258–269
Jacobson, G.R.; Schaffer, M.H.; Stark, G.R.; Vanaman, T.C. (1973): Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J. Biol. Chem. 248, 6583–6591
Kasarda, D.D.; Autran, J.C.; Lew, E.J-L.; Nimmo, C.C.; Shewry, P.R. (1982): N-terminal amino acid sequences of ω-gliadin and ω-secalins: implications for the evolution of prolamin genes. Biochim. Biophys. Acta. (subm. for publ.)
Kasarda, D.D.; Bernard, J.E.; Thomas, R.S. (1967) Reversible aggregation of α-gliadin to fibrils. Science 155, 203–205
Kasarda, D.D.; Bernardin, J.E.; Nimmo, C.C. (1976): Wheat proteins. In: Advances in Cereal Science and Technology I. (ed. Pomeranz, Y.), pp. 158–236. St. Paul, Minnesota: Am. Assoc. Cereal Chem.
Khan, K.; Bushuk, W. (1978): Glutenin: structure and functionality in breadmaking. Bakers Digest 52, 14–20
Khan, K.; Bushuk, W. (1979): Studies of glutenin XIII gel filtration, isoelectric focusing, and amino acid composition studies. Cereal Chem. 56, 505–512
Køic, B.; Ingversen, J.; Andersen, A.J.; Doll, H.; Eggum, B.O. (1976): Composition and nutritional quality of barley protein. In: Evaluation of Seed Protein Alterations by Mutation Breeding, pp. 55–61. Vienna: IAEA
Kulbe, K.D. (1974): Micropolyamide thin layer chromatography of phenylthiohydantoin amino acids (PTH) at subnanomolar level. A rapid microtechnique for simultaneous microsample identification after automated Edman degradations. Anal. Biochem. 59, 564–573
Lawrence, G.J.; Shepherd, K.W. (1980): Variation in glutenin protein subunits of wheat. Aust. J. Biol. Sci. 33, 221–233
Lawrence, G.J.; Shepherd, K.W. (1981a): Chromosomal location of genes controlling seed proteins in species related to wheat. Theor. Appl. Genet. 59, 25–31
Lawrence, G.J.; Shepherd, K.W. (1981b): Inheritance of glutenin protein subunits of wheat. Theor. Appl. Genet. 60, 333–337
Lee, J.C.; Timasheff, S.N. (1979): Calculations of protein partial specific volumes. In: Methods in enzymology, vol. 61, (eds. Hirs, C.H.W.; Timasheff, J.S.), pp. 49–57. New York: Acad. Press
Meredith, O.B.; Wren, J.J. (1966): Determination of molecular weight distribution in wheat flour proteins by extraction and gel filtration in a dissociating medium. Cereal Chem. 43, 169–186
Miflin, B.J.; Burgess, S.R.; Shewry, P.R. (1981): The development of protein bodies in the storage tissues of seeds. Subcellular separations of homogenates of barley, maize and wheat endosperms and of pea cotyledons. J. exp. Bot. 32, 199–219
Miflin, B.J.; Byers, M.; Field, J.M.; Faulks, A.J. (1980): The isolation and characterization of proteins extracted from whole milled grain, gluten and developing protein bodies of wheat. Ann. Technol. Agric. 29, 133–147
Miflin, B.J.; Field, J.M.; Shewry, P.R. (1982): Cereal storage proteins and their effect on technological properties. In: Proc. Seed Protein Symp. Versailles
Miflin, B.J.; Shewry, P.R. (1977): An introduction to the extraction and characterization of barley and maize prolamins. In: Techniques for the Separation of Barley and Maize Seed Proteins (eds. Miflin, B.J.; Shewry, P.R.), pp. 13–21. Luxembourg: EEC
Miflin, B.J.; Shewry, P.R. (1979): The biology and biochemistry of cereal seed prolamins. In: Seed Protein Improvement in Cereals and Grain Legumes, Vol. 1, pp. 137–158. Vienna: IAEA
Payne, P.I.; Corfield, K.G. (1979): Subunit composition of wheat glutenin proteins, isolated by gel filtration in a dissociating medium. Planta 145, 83–88
Payne, P.I.; Corfield, K.G.; Blackman, J.A. (1979): Identification of a high molecular weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theor. Appl. Genet. 55, 153–159
Payne, P.I.; Corfield, K.G.; Holt, L.M.; Blackman, J.A. (1981a): Correlations between the inheritance of certain high molecular weight subunits of glutenin and breadmaking quality in progenies of six crosses of bread wheat. J. Sci. Food Agric. 32, 51–60
Payne, P.I.; Holt, L.M.; Law, C.N. (1981b): Structural and genetical studies of the high-molecular-weight subunits of wheat glutenin. Part 1. Allelic variation in subunits amongst varieties of wheat (Triticum aestivum). Theor. Appl. Genet. 60, 229–236
Payne, P.I.; Law, C.N.; Mudd, E.E. (1980): Control by homoeologous group 1 chromosomes of the high molecular weight subunits of glutenin, a major protein of wheat endosperm. Theor. Appl. Genet. 58, 113–120
Pisano, J.J., Bronzert, T.J., Brewer, H.B., Jr. (1972): Advances in gas Chromatographic analysis of amino acid phenyl- and methylthiohydantoins. Anal. Biochem. 45, 43–59
Roark, D.E.; Yphantis, D.A. (1969): Studies of self-associating systems by equilibrium ultracentrifugation. Ann. N.Y. Acad. Sci. 164, 245–278
Schmitt, J.M.; Svendsen, I. (1980): Amino acid sequence of hordein polypeptides. Carlsberg Res. Commun. 45, 143–148
Schulz, G.E.; Schirmer, R.H. (1979): Principles of Protein Structure. Berlin: Springer Verlag
Shewry, P.R.; Hill, J.M.; Pratt, H.M.; Leggatt, M.M.; Miflin, B.J. (1978): An evaluation of techniques for the extraction and separation of hordein and glutelin from barley seed and a comparison of the protein composition of Bomi and Risø 1508. J. exp. Bot. 29, 677–692
Shewry, P.R.; Autran, J.-C.; Nimmo, C.C.; Lew, E.J-L.; Kasarda, D.D. (1980a): N-terminal amino acid sequence homology of storage protein components from barley and a diploid wheat. Nature 286, 520–522
Shewry, P.R.; Field, J.M.; Kirkman, M.A.; Faulks, A.J.; Miflin, B.J. (1980b): The extraction, solubility and characterization of two groups of barley storage polypeptides. J. exp. Bot. 31, 393–407
Shewry, P.R.; Field, J.M.; Lew, E.J-L., Kasarda, D.D. (1982a): The purification and characterization of two groups of storage proteins (secalins) from rye (Secale cereale L.). J. exp. Bot. 33, 261–268
Shewry, P.R.; Lew, E.J-L., Kasarda, D.D. (1981): Structural homology of storage proteins coded by the Hor 1 locus of barley (Hordeum vulgare L.). Planta 153, 246–253
Shewry, P.R.; Miflin, B.J. (1982): Genes for the storage proteins of barley. Qualitas Plantarum (Plant Foods for Human Nutrition) (in press)
Shewry, P.R.; Parmar, S.; Miflin, B.J. (1982b): The extraction, separation and polymorphism of the prolamin storage proteins (secalins) of rye. Cereal Chem. (in press)
Wall, J.S. (1979): The role of wheat proteins in determining baking quality. In: Recent Advances in the Biochemistry of Cereals (eds. Laidman, D.L.; Wyn Jones, R.G.), pp. 275–311. London: Acad. Press
Winter, A.; Perlmutter, H.; Davies, H. (1975): Preparative flatbed electrofocusing in a granulated gel with the LKB 2117 Multiphor. LKB Application Note No. 198. Sweden: LKB Produkter AB
Yphantis, D.A. (1964): Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3, 297–317
Author information
Authors and Affiliations
Additional information
Communicated by R. Riley
Rights and permissions
About this article
Cite this article
Field, J.M., Shewry, P.R., Miflin, B.J. et al. The purification and characterization of homologous high molecular weight storage proteins from grain of wheat, rye and barley. Theoret. Appl. Genetics 62, 329–336 (1982). https://doi.org/10.1007/BF00275097
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00275097