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

Abstract

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

The Current State of the Golgi Proteomes

“…With plant Golgi antibodies then, as now, commercially unavailable, enzyme assays were the primary means of determining fraction composition. Profiling by enzyme assays was not sufficiently precise or efficient for tracking lower-abundance Golgi proteins amidst a relatively complex background of contaminants, although the distribution of enzyme activities reported by (Sandelius et al, 1986) are broadly consistent with later proteomic analyses. The first isolation of plant Golgi membranes has depended on both FFE and proteomic advances (Parsons and Heazlewood, unpublished data).…”

“…Plant homogenates were first subjected to FFE some decades ago (Kappler et al, 1986;Sandelius et al, 1986;Bardy et al, 1998) but these first forays demonstrated little potential for Golgi isolation. With plant Golgi antibodies then, as now, commercially unavailable, enzyme assays were the primary means of determining fraction composition.…”

Tonoplast Vesicles of Opposite Sidedness from Soybean Hypocotyls by Preparative Free-Flow Electrophoresis

“…The identification of the tonoplast through the different fractions obtained by free-flow electrophoresis of microsomal membranes was previously achieved by membrane thickness measurements and nitrate sensitive ATPase activity (25). The most electronegative fraction (fraction A) was found to consist of highly pure tonoplast vesicles.…”

“…a ATPase activity was measured in the presence of 0.02% Triton X-1 00, the level of inhibitors was as for Table I. glucan synthetase II (25). A similar spectrum of markers is not available for the vacuolar membranes of plant cells (3).…”

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers

“…Aqueous polymer two-phase partition and freeflow electrophoresis not only result in highly purified preparations but also discriminate between membrane vesicles of opposite orientation (Larsson et al, , 1988Sandelius et al, 1986;Canut et al, 1987). The uncritical use of single biochemical markers for assessing the purity of plasma membrane fractions has often been questioned and additional methods, such as surface labelling and EM observation, have been used in order to provide confirmatory evidence of plasma membrane purification (Boss & Ruesink, 1979 ;Bowman et al, 1981;Scarborough, 1975;Sandelius et al, 1986). Additionally, limited 'attention to markers of other intracellular organelles which may contaminate plasma membrane fractions has compromised the degree of purity of many preparations.…”