| Oligogalacturonides were labeled with biotin using a rapid and simple two-reaction protocol. In the first reaction biotin-x-hydrazide was coupled to C-1 of the reducing galacturonic acid residue by a hydrazone linkage. Carbohydrate-hydrazone linkages have been widely used to label biomolecules. However, we show that oligogalacturonide-hydrazone linkages are unstable. In the second reaction the hydrazone linkage was reduced forming a stable hydrazide. Electrospray mass spectrometry and 1H-NMR spectroscopic analysis confirmed the structure of HPAEC-purified biotin-derivatized oligogalacturonides. Biotin-derivatized oligogalacturonides were used for studies of the oligogalacturonide biological function.; The biological activity of reducing end-modified oligogalacturonides was quantitatified in four Nicotiana tabacum tissue culture bioassays. The derivatives tested had C-1 of their reducing-end covalently linked to a biotin hydrazide, covalently linked to tyramine, chemically reduced to a primary alcohol, or enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, elicit extracellular alkalinization by suspension-cultured N. tabacum cv Samsun cells, elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and elicit H2OL accumulation in the cv Xanthi cells. The derivatives each had reduced biological activity in all four bioassays, demonstrating that the reducing end is involved in recognition of oligogalacturonides in these bioassays. However, the degree of reduction in biological activity depended on the bioassay and the nature of the reducing end modification, suggesting that the oligogalacturonides are perceived differently in each bioassay.; The lipopolysaccharides (LPSs) from four R. etli mutants, one R. leguminosarum bv. trifolii mutant and from the R. etli parent strain were isolated by hot phenol/water (&phis;/W), and phenol/EDTA/triethylarnine (&phis;/EDTA/TEA) extraction. Over 18 different LPS extraction methods have been reported, and none is universally applicable. The LPS in the preparations studied here was quantified, and analyzed by deoxycholate polyacrylamide gel electrophoresis, and immunoblotting. The LPS yield from all the strains using &phis;/EDTA/TEA extraction was consistent (three-fold range), while the yields from &phis;/W extraction were variable (850 fold range). Overall, &phis;/EDTA/TEA extraction yielded more (but not all) forms of LPS. It was concluded that careful analysis of any LPS mutant requires more than one extraction method. |
