PURIFICATION OF PLASMA-MEMBRANES FROM LEAVES OF CONIFER AND DECIDUOUSTREE SPECIES BY PHASE PARTITIONING AND FREE-FLOW ELECTROPHORESIS

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 mo re difficult to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by bo th phase partitioning and free-flow electrophoresis from microsomal fr actions and identified on the basis of biochemical and in some cases m orphological and cytochemical markers. Electron micrographs revealed t hat membrane vesicles from Pinus sylvestris exhibited a very clear dar k-light-dark pattern and measurements of membrane thickness showed tha t it ranged from 6 to 10 nm. Most membranes were 8 nm thick and staine d with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes con sisted 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 fr actions, depending on plant species. 1,3-beta-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. Contamina tion by membranes of other origin was determined by analysis of membra ne markers: cytochrome c oxidase (EC 1.9.3.1) for mitochondria, inosin e diphosphatase (EC 3.6.1.6) for Golgi apparatus, cytochrome c reducta se (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. P lasma membrane characteristics were quite different depending on the s eason during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter.