Isoelectric Focusing of Oat Root Membranes

Abstract

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...

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

“…New methods have appeared exploiting differences in vesicle surface charges rather than differences in density, i.e. aqueous two-phase partitioning (Albertsson 1971) and isoelectric focusing (Steele and Stout 1986). For the simultaneous separation of tonoplast and plasma membrane, on a preparative scale, the only method to be described is that of free-flow electrophoresis Sandelius et al 1986).…”