II. 8. 'D' Hordein, a new group of prolamin storage proteins in barley.
P. R. Shewry, J. M. Field, S. Parmar and B. J. Miflin, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ, United Kingdom. "R"
Three groups of hordein polypeptides (called A, B and C hordein) have been purified and characterized previously. B and C hordein are the major storage proteins present in protein bodies and appear to be controlled by co-dominant alleles at linked structural loci, called Hor 2 and Hor 1, respectively, located on the short arm of chromosome 5 (see Shewry and Miflin, 1982). The third group, A hordein, is a minor component and does not appear to have a storage function (see Salcedo et al., 1980).
Lawrence and Shepherd (1981) recently reported that an additional high molecular weight storage protein was coded for by genes on the long arm of chromosome 5. We have now purified this component from cv. Sundance and have called it D hordein. SDS-PAGE of the preparation shows a single band with an apparent mol. wt. of about 105,000, but the molecular weight by sedimentation equilibrium ultracentrifugation is considerably lower - 54,700. Isoelectric focusing showed the presence of a number of polypeptides. Its amino acid composition is similar to that of other prolamins in that it contains large amounts of glutamate + glutamine (30 mole %) and only traces of lysine (0.8 mole %). However, the content of proline is relatively low (11.4 mole %) and that of glycine unusually high (13.6 mole %). It appears to be present in protein bodies prepared from developing endosperms (Miflin et al., 1981) which confirms that it is a storage protein.
D hordein is poorly extracted by aqueous alcohols alone, and although extraction is improved by the addition of 2-mercaptoethanol (l to 2% v/v) and acetic acid (1% v/v) it is still not complete. Consequently, it is difficult to accurately determine the total amount present in the tissue. However, determinations based on scanning of SDS-PAGE separations of efficiently extracted fractions indicate that it accounts for l to 4% of the total hordein fraction.
SDS-PAGE analyses of hordein fractions from a large number of European cultivars has shown that they all have a single D hordein band with identical or closely similar mobility. We have, however, shown the presence of genetic variability in lines from the USDA World Barley collection, some having a single band with different mobility and while others have two bands (Shewry and Miflin, 1982).
We are at present mapping the structural genes for D hordein in collaboration with R. A. Finch, PBI, Cambridge.
Lawrence, G. J. and K. W. Shepherd. 1981. Chromosomal location of genes controlling seed proteins in species related to wheat. Theor. Appl. Genet. 59:25-31.
Miflin, B. J., S. R. Burgess and P. R. Shewry. 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.
Salcedo, G., R. Sanchez-Monge, A. Argamenteria and C. Aragoncillo. 1980. The A-hordeins as a group of salt-soluble hydrophobic proteins. P1. Sci. Lett. 19:109-119.
Shewry, P. R. and B. J. Miflin. (1982) Genes for the storage proteins of barley. Qualitas Plantarum (Plant Foods for Human Nutrition) (In Press)
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