Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H⁺-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.     

Carbonic anhydrase in the plasma membranes from leaves of C3 and C4 plants

Plasma membranes were isolated from green leaves of maize ( Zea mays ), spinach ( Spinacia oleracea ), Setaria viridis and wheat ( Triticum aestivum cv. Omase) by aqueous two-phase partitioning. Carbonic anhydrase activity was detected in these membranes. The activity was inhibited by specific inhibitors for carbonic anhydrase, acetazolamide and ethoxyzolamide. The carbonic anhydrase activity was markedly enhanced by the addition of Triton X-100 to the plasma membranes. The highest activity was obtained in the presence of 0.015% detergent. The activity was scarcely affected when the plasma membrane vesicles were treated with proteinase K, but largely inactivated by the protease after treating the membranes with Triton X-100. These results indicate that carbonic anhydrase faces the cytoplasmic side of the membrane since plasma membranes purified by aqueous two-phase partitioning are tightly sealed vesicles of right side-out orientation (apoplastic side-out). With leaves of C₄ plants, 20 to 60% of the total carbonic anhydrase activity was found in the microsomal fraction. By contrast, only 1 to 3% of the activity was found in the microsomal fraction from leaves of C₃ plants. Western blot analysis showed that a polypeptide in the spinach plasma membrane cross-reacted with an antiserum raised against spinach chloroplast carbonic anhydrase, and that the molecular mass of the plasma membrane enzyme was higher than that of the chloroplast carbonic anhydrase (28 and 26 kDa, respectively). This indicates the presence of different molecular species of carbonic anhydrase in the chloroplast and the plasma membrane.     

Preparation of mammalian plasma membranes by aqueous two-phase partition

Plasma membranes are readily purified from crude mixtures by the technique of aqueous two-phase partition. This procedure has been used widely to prepare plasma membrane fractions, highly purified, from both green and dark-grown plant materials. Only recently, however, has the method been applied to animal cells and tissues to supplant previous protocols where preparative sucrose and other gradient procedures were employed. The method based on aqueous two-phase partition, is rapid, reproducible and facile. It is especially useful for tissue culture cells since gradient methods often are complicated by alterations in plasma membrane density with different culture conditions and the presence of extensive cytoskeleton-membrane interactions. Homogenates prepared either in dilute 1 mM bicarbonate or isotonic sucrose are first centrifuged to concentrate the plasma membrane vesicles. The concentrated membranes are then combined with a mixture of dextran and polyethylene glycol that will of itself spontaneously separate into a polyethylene glycol-rich upper phase and a dextran-rich lower phase. The mixture is usually centrifuged to accelerate phase separation. The plasma membranes enter the upper, polyethylene glycol-rich phase, whereas contaminating membranes remain with the dextran of the lower phase. The yield of plasma membranes is 20% or more of those present in homogenates and the fraction purity is 90% or greater.     

Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.     

两相法分离地被菊叶片质膜的研究

以地被菊(Dendranthema×grandiflorum Kitamura)叶片为材料,通过水溶性聚合物Dextran T-500和PEG 3350所构成的两相分配体系制备质膜。在一定盐浓度（5 mmol.L-1 NaCl）下选用5种不同的聚合物浓度（5.8%、6.0%、6.2%、6.4%、6.6%,W/W）,研究了地被菊叶片质膜在两相体系中的分配情况,在此基础上进一步研究了不同盐浓度（2、4、5、10、20 mmol.L-1 NaCl）对地被菊叶片质膜的纯度及蛋白产率的影响。标志酶鉴定及磷钨酸染色电镜检测的结果表明,地被菊叶片选用6.2%（W/W）聚合物浓度和7 mmol.L-1 NaCl组成的两相分配体系可获得较高纯度的密实正向型质膜囊泡。     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction `E,' was pure plasma membrane. The other, more electronegative and designated fraction `C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.     

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 μU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristics responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.     

Phosphatidylinositol specific phospholipase C of plant stems : membrane associated activity concentrated in plasma membranes

A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1.4.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-1-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all major cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by two-phase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium.     

Use of a concanavalin A polymer to isolate right side-out vesicles of purified plasma membranes from eukariotic cells

We have purified two plasma membrane populations using a Concanavalin A polymer. It was assumed that vesicles retained by the polymer were right side-out, whereas vesicles not retained were inside-out. 5′-nucleotidase and (Na⁺ + K⁺) stimulated Mg⁺⁺ ATPase activities were at least two fold higher in inside-out than in right side-out vesicles, though recovered total activity was about 80 % for both enzymes together. Moreover, Concanavalin A modified 5′-nucleotidase activity of right side-out vesicles according to the dose used.     

Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system : on the use of light-induced cytochrome B reduction as a marker for the plasma membrane

Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.

The plasma membranes from both organs were enriched in K⁺-stimulated Mg²⁺-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.

The plasma membranes were also enriched in a light-reducible b-cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane.

     

Surface properties of right side-out plasma membrane vesicles isolated from barley roots and leaves

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K⁺,Mg²⁺-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic—about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being −29 mC·m⁻² for the leaf plasma membrane and only −19 mC·m⁻² for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca²⁺ and Mg²⁺ bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca²⁺ bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni²⁺ could bind to the root vesicles as indicated by the effect of Ni²⁺ on 9-aminoacridine fluorescence, and by the binding of ⁶³Ni²⁺ (44 nanomoles bound per milligram protein) at 100 micromolar NiCl₂.     

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

The Isolation of Plasma Membrane from Vicia faba Leaves and Pollen Tubes of Luffs cylindrica by the Two-Polymer Phase System

A method for the isolation of plasma membrane enriched fraction from plant sources is described. An aqueous two-phase system is suitable for the isolation of plasma membrane. Purified plasma membrane fractions have been prepared from Vicia faba leaves and pollen tubes of Luffa cylindrica. The determination of marker enzymes shows that the plasma membrane has a high affinity for the polyethylene glycol-rich upper phase, whereas those membranes from mitochondria, chloroplasts and other organelles prefer the dextran-rich lower phase and the interface. The plasma membrane was identified with the aid of silicotungstic acid-chromic acid staining. In the upper phase there are mainly stained elliptical vesicles, whereas in the lower phase there are many differnet kind of unstained vesicles. This result is coincident with the marker enzyme examination. The two-phase method for the isolation of plasma membrane may be widely applied not only to nongreen tissues but also to green tissues of plants.     

Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean

Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.     

Separation of dense, polysaccharide-containing vesicles from secreting, cultured oat cells. Characterization of a putative secretory vesicle fracton

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⁺, Mg²⁺-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.