Gibeaut DM, Carpita NC Synthesis of (1-3),(1-4)-β-D-glucan in the golgi apparatus of maize coleoptiles Proceedings of the National Academy of Sciences of the USA90 (1993)
3850-3854
NCBI PubMed ID:8483902 Publication DOI:10.1073/pnas.90.9.3850 Journal NLM ID:7505876 Publisher: National Academy of Sciences Institutions: Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155
Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1→3), (1→4)-β-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa and was separated from a much larger (1→3)-β-D-glucan (callose) by gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were separated by HPLC and GLC. The trisaccharide β-D-Glcp-(1→4)-β-D-Glcp-(1→3)-D-Glc, the tetrasaccharide [β-D-Glcp-(1→4)]2-β-D-Glcp-(1→3)-D-Glc, and longer cellodextrin-(1→3)-D-Glc oligosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhanced synthesis of MG, presumably by removing inhibitory compounds. The Golgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was restored by addition of either MgCl2 or, to a lesser extent, MnCl2. Synthesis continued for well over 1 h; addition of KOH to raise the pH from 7.2 to 8.0 during the reaction increased the rate of synthesis, which indicates that a transmembrane pH gradient may facilitate synthesis of MG.
Gibeaut DM, Carpita NC Synthesis of (1-3),(1-4)-β-D-glucan in the golgi apparatus of maize coleoptiles Proceedings of the National Academy of Sciences of the USA90 (1993)
3850-3854
NCBI PubMed ID:8483902 Publication DOI:10.1073/pnas.90.9.3850 Journal NLM ID:7505876 Publisher: National Academy of Sciences Institutions: Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155
Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1→3), (1→4)-β-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa and was separated from a much larger (1→3)-β-D-glucan (callose) by gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were separated by HPLC and GLC. The trisaccharide β-D-Glcp-(1→4)-β-D-Glcp-(1→3)-D-Glc, the tetrasaccharide [β-D-Glcp-(1→4)]2-β-D-Glcp-(1→3)-D-Glc, and longer cellodextrin-(1→3)-D-Glc oligosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhanced synthesis of MG, presumably by removing inhibitory compounds. The Golgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was restored by addition of either MgCl2 or, to a lesser extent, MnCl2. Synthesis continued for well over 1 h; addition of KOH to raise the pH from 7.2 to 8.0 during the reaction increased the rate of synthesis, which indicates that a transmembrane pH gradient may facilitate synthesis of MG.
Gibeaut DM, Carpita NC Synthesis of (1-3),(1-4)-β-D-glucan in the golgi apparatus of maize coleoptiles Proceedings of the National Academy of Sciences of the USA90 (1993)
3850-3854
NCBI PubMed ID:8483902 Publication DOI:10.1073/pnas.90.9.3850 Journal NLM ID:7505876 Publisher: National Academy of Sciences Institutions: Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155
Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1→3), (1→4)-β-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa and was separated from a much larger (1→3)-β-D-glucan (callose) by gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were separated by HPLC and GLC. The trisaccharide β-D-Glcp-(1→4)-β-D-Glcp-(1→3)-D-Glc, the tetrasaccharide [β-D-Glcp-(1→4)]2-β-D-Glcp-(1→3)-D-Glc, and longer cellodextrin-(1→3)-D-Glc oligosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhanced synthesis of MG, presumably by removing inhibitory compounds. The Golgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was restored by addition of either MgCl2 or, to a lesser extent, MnCl2. Synthesis continued for well over 1 h; addition of KOH to raise the pH from 7.2 to 8.0 during the reaction increased the rate of synthesis, which indicates that a transmembrane pH gradient may facilitate synthesis of MG.
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