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Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling

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Abstract

The preparation of a phenylalanine analogue containing an azido group and its incorporation into dipeptides is described. Peptides modified in this way are taken up into barley (Hordeum vulgare L.) scutella via the previously characterized peptide-transport system. Photoactivation of modified peptides in the presence of isolated scutella resulted in irreversible inhibition of peptide uptake in a concentration-dependent manner. Transport of other solutes which share a common mechanism of energy coupling, but which are transported via distinct carriers, was not inhibited after photo-derivatization of scutella with the modified peptides. Derivatization of isolated scutellar tissue with a 14C-labelled peptide analogue, resulted in incorporation of label into two proteins of Mr = 54000 and 41000. Scutellar tissue from early-germinating seeds, which do not show active peptide uptake, did not incorporate label into these polypeptides. It is concluded that these proteins are components of the barley peptide-transport system.

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Abbreviations

Ala:

alanine

Gly:

glycine

PAGE:

polyacrylamide gel electrophoresis

Phe:

phenylalanine

Pro:

proline

SDS:

sodium dodecyl sulphate

References

  1. Barrett, A.J., Kembhavi, A.A., Brown, M.A., Kirschke, H., Knight, C.G., Tamai, M., Hanada, K. (1982) l-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. 102, 189–198

  2. Bayley, H., Knowles, J.R. (1977) Photoaffinity labeling. Methods Enzymol 46, 69–114

  3. Dolphin, D., Wick, A. (1977) Tabulation of infra-red spectral data. Wiley-Interscience, Wiley & Sons, New York

  4. Enari, T.M., Mikola., J. (1977) Peptidases in germinating barley grain: properties, localization and possible functions. In: Peptide transport and hydrolysis (Ciba Foundation Symposium 50, N.S.) pp 335–352, Elsevier/Excerpta Medica/North Holland, Amsterdam

  5. Enari, T.M., Sopanen, T. (1986) Mobilization of endospermal reserves during the germination of barley. J. Inst. Brew. 92, 25–31

  6. Fincher, G.B. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 305–346

  7. Gallet, O., Lemoine, R., Earsson, C., Delrot, S. (1989) The sucrose carrier of the plant plasma membrane. I. Differential affinity labeling. Biochim. Biophys. Acta 978, 56–64

  8. Hancock, K., Tsang, V.C.W. (1983) India ink staining of proteins on nitrocellulose paper. Analyt. Biochem. 133, 157–162

  9. Heukeshoven, J., Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103–112

  10. Higgins, C.F., Payne, J.W. (1977) Characterization of active peptide transport by germinating barley embryos: effects of pH and metabolic inhibitors. Planta 136, 71–76

  11. Higgins, C.F., Payne, J.W. (1978a) Peptide transport by germinating barley embryos: evidence for a single common carrier for di and oligopeptides. Planta 138, 217–221

  12. Higgins, C.F., Payne, J.W. (1978b) Stereospecificity of peptide transport by germinating barley embryos. Planta 142, 299–305

  13. Higgins, C.F., Payne, J.W. (1980) Transport and utilization of amino acids and peptides by higher plants. In: Microorganisms and nitrogen sources, pp. 609–639, Payne, J.W., ed., John Wiley & Sons, New York

  14. Higgins, C.F., Payne, J.W. (1981) The peptide pools of germinating barley grains: relation to hydrolysis and transport of storage proteins. Plant Physiol. 67, 785–792

  15. Hurkman, W.J., Tanaka, C.K. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806

  16. Kramer, W. (1987) Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush border membrane vesicles by photoaffinity labeling. Biochim. Biophys. Acta 905, 65–74

  17. Kramer, W., Dechent, C., Girbig, F., Gutjahr, U., Neubauer, H. (1990a) Intestinal uptake of dipeptides and β-lactam antibiotics. I. The intestinal uptake system for dipeptides and β-lactam antibiotics is not part of a brush border membrane peptidase. Biochim. Biophys. Acta 1030, 41–49

  18. Kramer, W., Gutjahr, U., Girbig, F., Leipe, I. (1990b) Intestinal absorption of dipeptides and β-lactam antibiotics. II. Purification of the binding protein for dipeptides and β-lactam antibiotics from rabbit small intestinal brush border membranes. Biochim. Biophys. Acta 1030, 50–59

  19. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

  20. Mikola, L., Mikola, J. (1980) Mobilization of proline in the starchy endosperm of germinating barley grain. Planta 149, 149–154

  21. Noren, O., Sjostrom, H., Larsen, P. (1983) Na+-dependent uptake of 4-azidophenylalanine by pig intestinal microvillus vesicles. Interaction with neutral amino acid uptake and labelling pattern. Eur. J. Biochem. 134, 71–76

  22. Payne, J.W., Walker-Smith, D.J. (1987) Isolation and identification of proteins from the peptide-transport carrier in the scutellum of germinating barley (Hordeum vulgare L.) embryos. Planta 170, 263–271

  23. Rausch, T., Raszeja-Specht, A., Koepsell, H. (1989) Identification of a 75,000 component of the H+/D-glucose transporter from Zea mays with monoclonal antibodies directed against the mammalian Na+/D-glucose transporter. Biochim. Biophys. Acta 985, 133–138

  24. Rich, D.H., Bernatowicz, M.S., Agarwal, N.S., Kawai, M., Salituro, F.G. (1985) Inhibition of aspartic proteases by pepstatin and 3-methylstatin derivatives of pepstatin. Evidence for collected substrate enzyme inhibition. Biochemistry 24, 3165–3173

  25. Ripp, K.G., Vitanen, P.V., Francheschi, V.R. (1988) Identification of a membrane protein associated with sucrose transport into cells of developing soybean cotyledons. Plant Physiol. 88, 1435–1445

  26. Salmenkallio, M., Sopanen, T. (1989) Amino acid and peptide uptake in the scutella of germinating grains of barley, wheat, rice and maize. Plant Physiol. 89, 1285–1291

  27. Schlichter, D., Bajorat, K.-H. (1990) Photoaffinity labelling of membrane proteins involved in amino acid transport in sea anemones. Comp. Biochem. Physiol. 95A, 559–565

  28. Sopanen, T. (1984) Uptake of peptides and amino acids by the scutellum of germinating barley grain. In: Membrane transport in plants, pp. 365–370, Cram, W.J., Janacek, K., Rybova, R., Sigler, K., eds. Academia, Praha

  29. Sopanen, T., Burston, D., Taylor, E., Matthews, D.M. (1978) Uptake of glycylglycine by the scutellum of germinating barley grain. Plant Physiol. 61, 630–633

  30. Sopanen, T., Sinervo, T., Ahokas, H. (1985) Uptake of amino acids and peptides by developing barley embryos. Physiol. Plant. 65, 347–351

  31. Staros, J.V., Knowles, J.R. (1978) Photoaffinity inhibition of dipeptide transport in Escherichia coli. Biochemistry, 17, 3321–3325

  32. Walker-Smith, D.J., Payne, J.W. (1983) Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein. FEBS Lett. 160, 25–30

  33. Walker-Smith, D.J., Payne, J.W. (1984a) Characteristics of the active transport of peptides and amino acids by germinating barley embryos. Planta 162, 159–165

  34. Walker-Smith, D.J., Payne, J.W. (1984b) Characteristics of the protein carrier of the peptide transport system in the scutellum of germinating barley embryos. Planta 162, 166–173

  35. Walker-Smith, D.J., Payne, J.W. (1985) Synthesis of the peptide transport carrier of barley scutellum during the early stages of germination. Planta 164, 550–556

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Author information

Correspondence to J. W. Payne.

Additional information

This work was supported by a grant from the Agricultural and Food Research Council.

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Hardy, D.J., Payne, J.W. Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling. Planta 186, 44–51 (1991). https://doi.org/10.1007/BF00201496

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Key words

  • Embryo (peptide carrier)
  • Hordeum (embryo, peptide transport)
  • Peptide transport
  • Photoaffinity probe