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Separation of dense, polysaccharide‐containing vesicles from secreting, cultured oat cells. Characterization of a putative secretory vesicle fracton
Author(s) -
Racusen Richard H.
Publication year - 1988
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1988.tb02048.x
Subject(s) - vesicle , golgi apparatus , membrane , biochemistry , chromatography , sonication , chemistry , polysaccharide , fractionation , biology , endoplasmic reticulum
Suspension cultured oat ( Avena sativa L. cv. Garry) cells, which secrete polysaccharides into the medium, were used as starting material for analyses of Golgi‐derived vesicle membranes and plasma membranes isolated during cell fractionation. Vesicles collected by a procedure employing ultrafiltration followed by ultracentrifugation into a sucrose step gradient exhibited an equilibrium density of 1.27 g cm −3 when run on continuous sucrose gradients, a feature which is most likely attributable to the high concentration of enclosed polysaccharides. Brief sonication lowered the density of these vesicles to about 1.15 g cm −3 , as judged from the coincidence of the protein peak and the marker enzymes for Golgi [Triton‐stimulated UDPase, cold‐storage IDPase (EC 3.6.1.6)] and plasma membrane [vanadate‐inhibited K + , Mg 2+ ‐ATPase (EC 3.6.1.3)]. Sonication of these vesicles also greatly diminished the amount of detectable polysaccharide observed in a colorimetric assay for sugars. Fractionation of a plasma membrane‐enriched preparation from these cells on continuous sucrose gradients showed the major protein peak and the peak activity for the plasma membrane marker at 1.17 g cm −3 , however, there was also significant overlap with a mitochondrial [cytochrome c oxidase (EC 1.9.3.1)] peak at 1.18 g cm −3 , Smaller peaks of the Golgi markers were seen at 1.14 g cm −3 . Analyses of marker enzymes for ER and mitochondria (EC 1.6.99.3) showed little contamination of the membranes of presumptive secretory vesicles from these sources, and the lack of significant vanadate‐insensitive ATPase activity in the density range from 1.13–1.18 g cm −3 in either fractionation scheme suggests that these membranes do not include material from the tonoplast. The coincidence of markers for Golgi and plasma membrane with from the tonoplast. The coincidence of markers for Golgi and plasma membrane with the membranes of sonicated, dense vesicles, at a density slightly lower than that of plasma membranes prepared from the same cells, supports the possibility that membranes en route to the plasma membrane are incompletely differentiated.