A micro-scale method for determining relative metal-binding affinities of proteins.

This article describes a quick and easy method for determining relative binding affinities between proteins and metal ions. The method is based on separating unbound metal ions from metal ions bound to protein by ultrafiltration using microcentrifuge ultrafiltration units. Bovine serum albumin (BSA) was used as the test protein and the relative affinity towards divalent metal ions was found to be Cu2+ > Zn2+ > Cd2+ > Pb2+ > Ni2+ > Co2+, which corresponds to the relative orders reported in the literature.

A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata.

A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45-120 kDa on SDS-PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O- and N-glycosidic linkages with the majority of the carbohydrate being O-linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.

A putative O-methyltransferase from barley is induced by fungal pathogens and UV light.

A cDNA clone, pBH72-F1 (F1), was isolated from a cDNA library prepared from barley leaves 72 h after inoculation with Erysiphe graminis f.sp. hordei. The 1388 bp nucleotide sequence of pBH72-F1 contains an open reading frame encoding a 42.3 kDa polypeptide of 390 amino acids which shows sequence similarity to O-methyltransferases (OMTs) from different plant species; the highest identity (41%) was observed with a putative OMT expressed in roots of maize. A phylogenetic analysis shows that the barley and maize sequences are distinctly different from the ortho-diphenol-OMTs involved in lignin formation. A putative S-adenosyl-L-methionine-binding motif (KELVDDSITN) determined for a rabbit protein-carboxyl OMT is partially conserved in the encoded amino acid sequence. Genomic Southern blot hybridization shows that pBH72-F1 probably represents a single copy gene. The F1 clone corresponds to a gene transcript exhibiting a relatively late accumulation in mildew-infected barley leaves compared to other pathogen-induced transcripts, such as transcripts encoding PR proteins, a peroxidase, and transcripts homologous to a maize caffeic acid OMT. No transcript was detected in plants exhibiting papilla resistance at time points when resistance is thought to be manifested. The atypical transcript accumulation pattern for F1 was also observed after infection by other pathogens and after UV-light treatment.

Purification, characterization, and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.

Partial amino acid sequences of two proteins, purified from barley leaves reacting hypersensitively to the powdery mildew fungus, showed a high degree of amino acid identity to the PR-1 proteins originally described in tobacco. The proteins, subsequently designated HvPR-1a and HvPR-1b, show apparent pI values of approximately 10.5 and 11, respectively and apparent M(r) 15,000. Independently, differential screening of a cDNA library prepared from barley leaves, exhibiting a compatible interaction with the powdery mildew fungus, resulted in isolation of cDNA species representing two PR-1 homologs. With the exception of one amino acid, the partial amino acid sequences of HvPR-1a and HvPR-1b are identical to internal sequences of the polypeptides derived from the two cDNA species. These derived polypeptides are each 164 amino acids long and both have putative N-terminal leader sequences of 24 amino acids. That these proposed leader sequences are functional is indicated by the observed occurrence of both proteins in the intercellular fluid. The proposed mature proteins (calculated M(r) 14,490 and 15,204) share 91% identical amino acids with each other and 56 to 74% with other PR-1 proteins. Northern blot hybridization and immunoblotting, respectively, show that both transcripts and both proteins accumulate following inoculation of susceptible and hypersensitivity resistant barley leaves with the powdery mildew fungus.