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Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free‐flow electrophoresis
Author(s) -
Toll Elena,
Castillo Federico J.,
Crespi Pierre,
Crèvecoeur Michèle,
Greppin Hubert
Publication year - 1995
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1995.tb00855.x
Subject(s) - membrane , chemistry , chromatography , free flow electrophoresis , biochemistry , picea abies , atpase , botany , biology , enzyme , polyacrylamide gel electrophoresis , gel electrophoresis of proteins
Purified plasma membrane fractions were obtained from leaves of Picea abies L., Pinus sylvestris L., Fagus sylvatica L. and Quercus robur L., whereas plasma membranes from Pinus halepensis Mill, proved to be more difficult 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 microsomal 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 pattern 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 vanadate‐sensitive ATPase (EC 3.6.1.3) activity. The specific activity in the upper phase (U 2 ) fraction was 10–25 times higher than those in the lower phase and microsomal fractions, depending on plant species. 1,3‐β‐glucan synthase II (EC 2.4.1.3), another putative plasma membrane marker, was not detected in the plasma membrane‐enriched fractions of conifer needles and showed a very low specific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome c 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.