Orientation of cell wall β-glucan synthases in plasma membrane vesicles

Abstract

In cell-free experiments with membranes of tobacco callus UDP-Glc incorporation into β-glucans was influenced by UDP. With UDP in the assay, β-glucan formation was inhibited and almost completely vanished at concentrations higher than 10 mM. Similar levels of inhibition were achieved with ADP and GDP, whereas the respective nucleotide-triphosphates inhibited to a higher and the nucleotide-monophosphates to a lower degree. The inhibition with UDP was shown to be non-competitive. UDP increased the β-glucan formation by variable degrees, when it was enclosed within membrane vesicles. The stimulation of β-glucan biosynthesis with enclosed UDP occurred mainly in inside out (ISO) plasma membrane vesicles. UDP in the assay inhibited the β-glucan formation as well in right side out (RSO) membrane vesicles as in ISO vesicles. Inhibitors of phospholipid glycosylation like amphomycin, tunicamycin and bacitracin inhibited β-glucan formation in the presence of 0.1% digitonin. Bacitracin without digitonin the in vitro β-glucan biosynthesis. The kinetics for inhibition by amphomycin and bacitracin were compared with kinetic parameters of different concentrations of digitonin. The results were discussed with respect to the two possibilities of enzyme/substrate interactions.