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

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 (14)C-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.

Nitrate transporters and peptide transporters

“…The barley scutellar peptide transporter, HvPTR1, is the best characterized plant PTR, as peptide transport in germinating barley grain has been extensively studied using biochemical approaches [18,47,48]. Unlike AtPTRs, which are expressed in almost all tissues, expression of HvPTR1 is highly tissue-and developmental stage-specific, with transcripts being detected in scutellar epithelial cells during germination [18].…”

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination

“…Both thiol affinity labelling, using either 4N 3 )Phe, the latter also labelling a third polypeptide of 54 kDa (Hardy and Payne, 1991;Payne and Walker-Smith, 1987). The relationship between the affinity labelled polypeptides and HvPTR1 awaits further experimental analysis, in particular immunological detection of the peptide transporter in scutellar extracts.…”

“…Substrate screening was used in an approach to specifically label proteins involved in peptide transport with [ 203 Hg]pCMBS, which identified two polypeptides of µ 66 kDa and 42 kDa as putative components of the scutellar peptide transporter (Payne and Walker-Smith, 1987). Photoaffinity labelling with [ 14 C]Ala-(2NO 2 , 4N 3 )Phe identified polypeptides of 54 kDa and 41 kDa as peptide binding proteins (Hardy and Payne, 1991).…”

Gibberellins: regulating genes and germination

“…In addition, amino acid and sugar transport to the embryo (e.g. Hardy & Payne, 1991 ;Bush, 1993), dissociation of endogenous hydrolase inhibitors (Halayko, Hill & Svensson, 1986) and solubilization of otherwise inaccessible hydrolase substrates (Hamabata et al, 1988) should all be favoured by an acidic environment. Thus coordination of extracellular pH and synthesis and secretion of hydrolases can be seen to be integral parts of GA action in the germinating grain.…”