Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free-flow electrophoresis

Abstract

Crevecoeur. M. and Greppin. H, 1995. Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free-flow electrophoresis. Purified piasma membrane fractions were obtained from leaves of Picea abies L,, Pinus sylvestris L,. Fagus sylvatica L. and Quercus robur L,, whereas plasma membranes from Pintjs halepensis Mill, proved to be more difficuh to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by both phase partitioning and free-flow electrophoresis from microsonnal fractions and identified on the basis of biochemical and in some cases morphological and cytochemical markers. Electron micrographs revealed that membrane vesicles from Pinus sylvestris exhibited a very clear dark-light-dark pattem and measurements of membrane thickness showed that it ranged from 6 to 10 nm. Most membranes were 8 nm thick and stained with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes consisted in the determination of the vanadaie-sensitive ATPase (EC 3,6,1,3) activity. The specific activity-in the upper phase (U2)fraction was 10-25 times higher than tliose in the lower phase and microsomal fractions, depending on plant species, 1.3-/y-glucan svnthase II (EC 2,4,1,3), another putative plasma membrane marker, was noi detected in the plasma membrane-enriched fractions of conifer needles and showed a very low speeific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome r oxidase (EC 1,9,3.1) for mitochondria, inosine diphosphatase (EC 3,6,1,6) for Golgi apparatus, cytochrome c reductase (EC 1,6.2.4) for endoplasmic reticulum. and pyrophosphatase (EC 3,6,1,1) for tonoplasts. The main, but relatively low contamination, was due to tonoplasts. as determined by the activity of pyrophosphatase. Plasma membrane characteristics were quite different depending on the season during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter,

Purification of plasma membranes from dry maize embryos

“…Under the electron microscope (Fig. 2), the vanadate-sensitive ATPase-enriched fraction exhibited an homogenous population of membrane vesicles similar to that of other plasma membrane preparations that have been previously described from other sources (Du Pont and Leonard 1980, Yoshida et al 1983, Canut et al 1988, Johansson et al 1995, Toll et al 1995.…”

“…Diacylglycerol acyl transferase (DAG-AT, EC Enzyme markers for tonoplast, mitochondria, plasma 2.3.1.20), a rough endopiasmic reticulum marker, was membranes (Ray 1979, Briskin et al 1987, Faraday and measured by incorporation of oleic acid from ''^C-oleoyl Spanswick 1992, Larsson et al 1994, Toll et al 1995, CoA to triacylglycerol by the method of Hosaka et al and endopiasmic reticulum (Hosaka et al 1977, Cao and(1977) with the following modifications: 150 | LI1 of a re-Huang 1986) were measured. action mixture consisting of 84 mM sucrose, 100 mM ATP hydrolysis by H"-ATPases from tonoplast (EC Tris/HCl, pH 7.4, 2 mM MgCl2, 400 |JM diacylglycerol, 3.6.1.3) (nitrate sensitive), mitochondria (EC 3.6.1.34) 300 pMCoA, 30|iMCoenzyme A [oleoyl-l-'^C ]-oleoyl (74 kBq per assay, 1.628 TBq moi"', DuPont NEN Research Products) and 0.2% (v/v) Tween 20 was incubated with 15 |ig of membrane protein for 25 min at 30°C.…”

On the response of pigments and antioxidants of Pinus halepensis seedlings to Mediterranean climatic factors and long‐term ozone exposure

“…For that reason, detergents were omitted in routine experiments. Special difficulties and interferences have been observed during the preparation of cellular extracts of Pinus halepensis (Toll et al, 1995), probably owing to the high content and peculiar compositions of resins and volatile substances in this Mediterranean conifer.…”

Isolation of cellular membranes from lignin‐producing tissues of Norway spruce and analysis of redox enzymes

“…Cells (200 g) were ground in liquid nitrogen to a fine powder with a pestle and mortar. The powder was allowed to thaw with gentle mixing in 500 ml of the homogenization buffer (modified from Toll et al ; Kaakinen, Finnish Forest Research Institute, personal communication) that contained 50 m M 3‐[ N ‐morpholino]propanesulfonic acid (MOPS)‐KOH, pH 7.5, 5 m M EDTA, 0.5 M sucrose, 1.5% (w/v) polyvinylpyrrolidone PVP K‐30 (Fluka, Pf‐Steinheim, Germany) and freshly‐added 5 m M dithiotreitol (DTT), 5 m M ascorbic acid, 4 m M cystein and 0.5 m M phenylmethylsulfonyl fluoride (PMSF). To remove cell debris the homogenate was filtered through a nylon cloth, and centrifuged for 10 min at 10 000 g at 4°C.…”

“…In this article, we have studied redox systems that are present in cellular membrane fractions isolated from two lignin‐producing materials: the extracellular lignin‐producing cultured cells (Simola et al ) and developing xylem of Norway spruce [ Picea abies (L.) Karst.]. As membrane isolation from conifers is more demanding than from angiosperms (Toll et al ), we present modifications that are essential to obtain cellular membranes from the phenolics‐rich Norway spruce tissues. We show that similarities exist in the membrane‐bound redox enzyme patterns in the two lignin‐producing tissues, and that there are redox systems present capable of transferring electrons from NAD(P)H to O 2 and thus forming O 2 •− .…”