Isoelectric focusing of plant cell membranes

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and KV-stimulated, vanadate-inhibited Mg2e ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH-and NADPHcytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-pnitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.With elongating plant cells, the total membrane surface contributed by tonoplast surrounding the central vacuole and the I

“…8-10, for review) or by aqueous two phase systems consisting of Dextran T 500 and PEG 3350 (13). Other techniques such as isoelectric focusing also have been applied (4).…”

mentioning

confidence: 99%

Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis

Penel²,

Auderset³

et al. 1986

“…The apparatus diagramed in Figure 1 is a simplified adaptation of a microanalytical IEF column described by Griffing and Quatrano (3) from an original design by Sherbet (10). Electrofocusing was conducted as previously described (3,10). All operations were conducted at 5°C.…”

Section: Methodsmentioning

confidence: 99%

“…Free-flow electrophoresis (9), aqueous two-phase partitioning (6,12,13), and IEF3 (3,10) have been used to separate membranes according to differences in their surfacecharge properties. Although effective at fractionating plant membranes (9), free-flow electrophoresis requires a major investment in equipment.…”

mentioning

confidence: 99%

Isoelectric Focusing of Oat Root Membranes

Steele

Stout²

1986

The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrowrange (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient.Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.Progress in plant membrane research has been hampered by the inability to obtain pure membrane preparations from plants. Most plant membrane isolation procedures have involved differential and sucrose density gradient centrifugation (4, 5), but a serious problem with this approach is cross-contamination of membrane fractions due to overlap in their densities (4).We have examined the possibility of purifying plant cell membranes on the basis of their surface-charge properties rather than their densities. Free-flow electrophoresis (9), aqueous two-phase partitioning (6, 12, 13), and IEF3 (3, 10) have been used to separate membranes according to differences in their surfacecharge properties. Although effective at fractionating plant membranes (9), free-flow electrophoresis requires a major investment in equipment. Both aqueous two-phase partitioning and microanalytical IEF are relatively inexpensive to perform (10, 12). We have chosen to investigate IEF to avoid two problems which are inherent to aqueous two-phase partitioning, namely, the apparent loss of membrane material and the exposure of membranes to PEG (12,13 plants.Here we report our preliminary findings regarding the feasibility of using IEF to obtain highly purified membranes from oat root tissue. MATERIALS AND METHODSPlant Tissue. Oat (Avena sativa var. Gerry) seedlings were grown in moist vermiculite at 280C in the dark. After 6 d, roots (10-15 cm in length) were harvested and stored (about 30 min) in ice-cold water prior to homogenization.Membrane Isolation. Roots were homogenized in mortar and pestle, and a crude membrane fraction was obtained as previously described (5, 1 1). The homogenization medium consisted of 250 mm sucrose, 1 mM EDTA, 25 mm MES-Tris (pH 7.5), 1 mM DTT, and 0.2 mM PMSF. After filtration through cheesecloth, the homogenate was centrifuged at 13,000g for 10 min. The 13,000g supernatant was centrifuged at 80,000g for 30 min to obtain a crude microsomal fraction. Crude membranes (13,000-80,000g pellet) were fractionated on a 2...

“…The apparatus was described by Griffing and Quatrano (6). Clinical grade dextran (average mol wt 80,000) was obtained from Sigma Chemical Co. BioLyte ampholytes, pH range 3 to 5, 4 to 6, and 5 to 7, were obtained from BioRad Laboratories.…”

Section: Methodsmentioning

confidence: 99%

“…The IEF technique used here to separate plant cell protoplasts is based on that developed by Griffing and Quatrano (6) to determine the surface charge on plant organelles. The distinction between EPM techniques and IEF techniques is that while EPM is the rate of movement of the cells in an electric field at a constant pH, the pl is the pH at which the cells in an electric field no longer move.…”

mentioning

confidence: 99%

Isoelectric Focusing of Plant Cell Protoplasts

Griffing

Cutler²,

Shargool³

et al. 1985

Self Cite

ABSTRACIThe surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pl). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 ± 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants.The zeta potential (a quantity related to surface charge) of plant protoplasts has been determined by measurements of the speed of protoplast movement in an electric field, their EPM' (7,15,18). Protoplasts derived from different tissue types and species were found to have different zeta potentials at constant pH, ranging (at pH 6) between -10 mv for mesophyll protoplasts from Vigna unguiictulata leaves to -36 mv for cortical protoplasts from Zea mays roots (15). Cell zeta potential has been correlated with several parameters such as viability (16) The IEF technique used here to separate plant cell protoplasts is based on that developed by Griffing and Quatrano (6) to determine the surface charge on plant organelles. The distinction between EPM techniques and IEF techniques is that while EPM is the rate of movement of the cells in an electric field at a constant pH, the pl is the pH at which the cells in an electric field no longer move. MATERIALS AND METHODSProtoplasts. Protoplasts were isolated from the leaves of periwinkle (Catharanthus roseus) plants, suspension culture cells of periwinkle cell line 916, and soybean (Glycine max) cell line SB-1 as described by Constabel (3).Material. The apparatus was described by Griffing and Quatrano (6). Clinical grade dextran (average mol wt 80,000) was obtained from Sigma Chemical Co. BioLyte ampholytes, pH range 3 to 5, 4 to 6, and 5 to 7, were obtained from BioRad Laboratories. All other chemicals were of the best grade commercially available.Electrofocusing Conditions. All gradients were prepared using equal volumes of solutions to be mixed, in a 50 ml gradient maker (Metaloglass Inc., Boston, MA). Low ionic strength acetate-containing gradients were prepared by mixing a high density (10% w/w) dextran solution containing 10 mm sodium acetate, 5 mm acetic acid, and 370 mm sorbitol with a low density (1% w/w) dextran solution containing 10 mM sodium acetate, 30 mM acetic acid, and 320 mm sorbitol. High ionic strength acetate-containing gradients were made up from a low density (1% dextran w/w) solution containing 50 mM sodium acetate, 100 mm acetic acid, and 100 mM sorbitol a...