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Journal of’General Microbiology (1 987), 133, 93-1 01. Printed in Great Britain
93
The Isolation of Plasma Membrane from the Diatom PhaeoductyIum
tricornutum Using an Aqueous Two-Polymer Phase System
By K . J . FLY”,*
H. OPIK AND P. J . SYRETT
Plant and Microbial Metabolism Research Group, School of Biological Sciences,
University College Swansea, Singleton Park, Swansea SA2 8PP, U K
(Received 3 July 1986; revised 23 August 1986)
Plasma membrane of the marine diatom Phaeodactylum tricornutum was purified by the
application of the microsomal fraction to an aqueous two-polymer phase system containing
5.7% (w/w) each of polyethylene glycol ( M , 3340) and dextran T500, plus 50 mM-NaC1 in a
Tris/maleate buffer (pH 7.3) with 500 mM-sorbitol. 5’-Nucleotidase, used as a marker for the
plasma membrane, partitioned differentially into the upper phase, and chlorophyll into the lower
phase. In sectioned intact cells only the plasma membrane and tonoplast stained with periodic
acid-chromic acid-phosphotungstic acid as viewed by electron microscopy. All of the
membranous material which partitioned into the upper phase of the two phase system stained
with this procedure, whilst much of the material in the lower phase did not. This result indicated
the presence of plasma membrane and/or tonoplast only in the upper phase; the increased
specific activity of 5’-nucleotidase indicated a 25-fold purification of plasma membrane in this
fraction compared to broken cells.
In order to differentiate between plasma membrane and tonoplast, vanadate-sensitive,
nitrate-insensitive, azide-insensitive, molybdate-insensitive K+,Mg2+-ATPasewas used as an
additional marker for plasma membrane, and acid phosphatase and nitrate-sensitive ATPase
were used as markers for tonoplast. Use of these markers indicated the presence of some
tonoplast membrane in the upper phase. It is concluded that the procedure used separates
plasma membrane almost completely from organelle membranes and partially from tonoplast.
INTRODUCTION
’
When cells of the marine diatom Phaeodactylum tricornutum are deprived of nitrogen in
conditions that allow them to photosynthesize, they develop the ability to take up various
nitrogenous compounds (Syrett et al., 1986). The addition of cycloheximide, an inhibitor of
protein synthesis, stops the development of these uptake systems (Flynn & Syrett, 1985).
However, it is not known if cycloheximide directly prevents the synthesis of proteins associated
with uptake, or if an activation of pre-existing transport systems is prevented by a lack of
necessary stimuli. Comparison of the protein composition of the plasma membranes (the most
likely location for proteins associated with uptake) from N-deplete and N-replete cells of P.
tricornutum may enable these possibilities to be distinguished.
The purification of plasma membrane from walled, pigmented plant cells using density
gradient centrifugation is complicated by contamination of the plasma membrane fraction by
chloroplast fragments (Leonard & Hodges, 1980; Yoshida et al., 1983). A method of separating
plasma membranes from other subcellular components has, however, been developed by
making use of the differential partition of membranes in a two-polymer phase system, as
Abbreviations: DCCD, N,N’-dicyclohexylcarbodiimide; DES, diethylstilboestrol ; 5’-NDase, 5’-nucleotidase;
NEM, N-ethylmaleimide; PACP, periodic acid-chromic acid-phosphotungstic acid; PEG, polyethylene glycol;
STM buffer, 500 mM-sorbitol, 15 mM-Tris/maleic acid (pH 7.3).
0001-3569 0 1987 SGM
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K . J . F L Y N N , H . OPIK AND P . J . SYRETT
pioneered by Albertsson (197 1). By the use of such systems considerable progress has now been
made in purifying plasma membrane from higher plants (Widell et al., 1982; Yoshida et al.,
1983; Wingstrand, 1985) and from yeast (Perez Cab0 et al., 1983).
The main criteria which are currently taken to indicate the presence of purified plasma
membranes from plant cells are that the vesicles should stain with periodic acid-chromic acidphosphotungstic acid (PACP) (Roland et al., 1972), and that a vanadate-sensitive, nitrateinsensitive, azide-insensitive, K+,Mg*+-ATPase with an optimum pH of 6-7 should be the
dominant ATPase (Leonard & Hodges, 1980; Goldfarb & Gradmann, 1983; Yoshida et al.,
19860). In addition, plasma membranes should not aggregate in response to low pH or high
Zn2+concentrations (Uemura & Yoshida, 1983). In Dictyostelium discoideum, the activity of a
plasma membrane bound 5'-nucleotidase has been used as a marker for the plasma membrane
(Gilkes & Weeks, 1977).A similar enzyme has been detected in P-deplete cells of P.tricornutum
and appears to be bound to the plasma membrane (Flynn et al., 1986).
This paper describes the first reported application of phase partition methods for the
purification of plasma membranes from a microalga.
METHODS
Growth and preparation of organism. Phaeodactylum tricornutum Bohlin (Culture Collection of Algae and
Protozoa, UK, strain 1052/6) was grown and prepared as described by Flynn et al. (1986).
Preparation of subcellular fractions. Cells were disrupted by shaking with glass beads (no. 10 ballotini), and
differentially centrifuged to yield a microsomal fraction (Flynn et al., 1986). The microsomal pellet was
resuspended in 500 mM-sorbitol, 15 mM-Tris/maleic acid, pH 7.3 (STM buffer), and pelleted by centrifugation at
156000g for 40 min at 5 "C. The pellet was resuspended in fresh STM buffer, and applied to a two-polymer phase
system.
The aqueous two-polymer phase system. The microsomal pellet from 4-5 g wet wt cells suspended in STM buffer
was applied to a 10 g (final wt) two phase system containing 5.7% (w/w) polyethylene glycol (PEG) M , 3340
(referred to as PEG 4000 in some sources), plus 5.7%(w/w) dextran T500. Stock solutions (2.5 g) containing 2243%
(w/w) of each polymer in STM buffer were added to 5 g sample in STM buffer. NaCl was added to the sample
(included in the 5 g) in order to give the required concentration in the final 10 g system. The system, at room
temperature, was mixed by 30 inversions of the tube, allowed to equilibrate at 0 "C (on ice) for 5 min, and then
mixed by a further 30 inversions. After partition at 0 "C (2-14 h), the upper phase was removed by pipette, the
material at the interface being included in the lower phase. Additional partitions were made if required by mixing
the upper phase with a freshly prepared lower phase.
Enzyme, protein and chlorophyll assays: efects of H + and Zn2+ concentration. 5'-Nucleotidase (EC 3 . 1 . 3 . 5; 5'NDase), acid phosphatase (EC 3 . 1 . 3 . 2 ) and ATPase (EC 3 . 6 . 1 . 3 ) activities were all measured in 100 mMTris/MES (pH range 5-9.5) or 100 mM-glycine/NaOH(pH range 8.5-10.5) containing 200 mM-NaC1,200 mM-KC1
8 mM-MgC12 and 2.5 mM substrate (sodium AMP, dicyclohexylammonium p-nitrophenylphosphate, or sodium
ATP). Samples (50 pl) from the phase separation were used, in some instances after dilution with STM buffer, to
give a concentration of 5-50 pg protein in 1ml final volume. Enzymes were assayed in 1.5 ml microtubes at 35 "C
for 10-30 min. At intervals, samples (50 or 100 pl) were taken for the determination of P, by modification of the
method of Lanzetta et al. (1979) as described by Flynn et al. (1986). Assays were done twice with further
duplication of individual P, measurements. After the final samples had been taken, the pH of the remaining assay
suspension was measured using a Russell CTWL pH probe. Protein was assayed using the BioRad microassay,
with bovine serum albumin as standard. Chlorophyll was assayed by the method of Parsons et al. (1984) after
extraction from a sample (50 pl) of the phase preparation by 1 ml acetone.
Effects of H+ and ZnZ+were measured by resuspending samples (100 pl) from the upper and lower phases in
900 pl STM buffer containing various concentrations of ZnC12, or in 900 pl 500 mM-sorbitol containing 10 mMsodium citrate buffer adjusted to various pH values and incubating at 20 "C for 30 min. Samples from the lower
phase were diluted in order to achieve an equal concentration of protein in comparison with upper phase fractions.
Membrane aggregation was measured by apparent absorbance at 510 nm against a sample at pH 7.25 (H+ series)
or in 0 mM-ZnC1, (Zn?+ series). All experiments were done on at least three separate occasions; representative
data are presented.
Electron microscopy. The upper and lower phase fractions were pelleted by centrifugation at 156000 g for 45 min
at 5 "C, resuspended in 50 mM-potassium phosphate buffer, pH 7.1, containing 13.5% (w/v) sucrose, and
repelleted as above. The pellets were fixed in 3% (v/v) glutaraldehyde in buffer containing 10% (w/v) sucrose for
1 h at 4-5 "C, followed by 1 h at room temperature. The pellets were washed in potassium phosphate buffer with
13.5%(w/v) sucrose using three changes over 45 min, and postfixed for 2 h in 2% (w/v) osmium tetroxide in buffer.
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Diatom plasma membrane
The material was dehydrated in an ethanol series and embedded in white acrylic resin, soft grade. Intact cells,
microsomal, and chloroplast and mitochondria1 fractions (Flynn et al., 1986) were prepared by the same
procedure, except that intact cells were embedded in medium grade resin.
Sections were cut on a LKB Ultrotome. For morphological examination, silver-coloured sections were mounted
on copper grids and stained with aqueous uranyl acetate and lead citrate. For PACP staining, sections with gold
interference colours were mounted on nickel grids; for uniformity of treatment, sections from membrane pellets
and intact cells were mounted on the same grids. A number of trials were run to optimize the staining procedure. In
the method finally adopted, sections were bleached for 60 min in 1% periodic acid and washed with 5 x 1 min
changes of distilled water. They were then stained for 10 min with 1% phosphotungstic acid in 10% (w/v) chromic
acid for 10 min, and again washed as after bleaching. When sections appeared to be unstable, they were stabilized
with a thin collodion film after staining. Specimens were examined in an AEI Corinth 275 electron microscope at
60 kV.
Chemicals. Fixatives and stains for electron microscopy were obtained from TAAB, the resin from London
Resin, and nickel grids from Agar Aids. Dextran T5OO was purchased from Pharmacia, and all other chemicals
were from BDH or Sigma.
RESULTS
After preliminary experiments in which both the M , and the concentration of the polymers
were varied, the system chosen to give an initial partitioning of all material into the upper phase
contained 5.7% (w/w) each of PEG 3340 and dextran T500, similar to the system used by
Yoshida et al. (1983).
Using the activity of 5’-NDase at pH 9.5 as a marker for the plasma membrane of P-deprived
cells (Flynn et al., 1986), a differential partitioning of the plasma membrane into the upper phase
with increasing concentrations of NaCl was observed (Fig. 1). As the NaCl concentration was
raised, the pigmented material and most of the protein partitioned into the lower phase. Over
60% of the 5’-NDase activity remained in the upper phase after the addition of 45 mM-NaC1,
resulting in a 6-fold increase in specific activity (Fig. 1). There was a 25-fold increase in specific
activity of 5’-NDase over that present in the broken cell suspension. The pH-activity profile of
the 5’-NDase isolated in the upper phase of a system with 50 mM-NaC1 was identical to that
previously measured in intact cells and in the whole microsomal fraction, with an optimum pH of
about 9.5 (cf. Flynn et al., 1986).
At this stage the material was examined by electron microscopy. In sections of intact cells,
PACP stained the plasma membrane and tonoplast (Fig. 2c). All attempts to achieve
- 8400
- 300
- 200
- 100
- 10
0
15
30
NaCl concn (mM)
45
Fig. 1. Effect of NaCl concentration on the partition of membrane associated with 5’-NDase and of
chloroplast fragments. The microsomal fraction of P-deplete cells was applied to a two-phase system
described in the text. Data are plotted as percentages of total protein (O),chlorophyll a (e),and 5‘NDase activity (A)
which partitioned into the upper phase. The specific activity of 5’-NDase in the
upper phase (I)
is also shown.
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K . J . F L Y N N , H . O P I K A N D P. J . S Y R E T T
Fig. 2. Electron micrographs of sectioned intact cells (bars 0.5 pm). (a) Uranyl acetate-lead citrate
stained. Cell at top shows chloroplast with pyrenoid; cell at bottom is cut through vacuole. (b)Periodic
acid bleached. All membranes completely bleached; lipid grey. (c) PACP stained. Stain in plasma
membrane and tonoplast (top, around granular vacuole) ; the pyrenoid (oval, left) indicates position of
chloroplast.
differential staining of these two membranes by varying staining times, varying washing times,
substitutions of silicotungstic acid for phosphotungstic acid and replacing chromic acid with
HCl, failed. There was also some staining in membranous material between the plasma
membrane and the cell wall. The specificity of PACP for some plant plasma membranes has
been questioned by Roland et al. (1972) and Nagahashi et al. (1978), but no staining was
observed in the nuclear membrane, mitochondria, Golgi apparatus, endoplasmic reticulum or
chloroplast envelope of P. tricornuturn. Thylakoids in intact cells stained very faintly, but in
chloroplast pellets, occasional more-dense staining occurred in swelling thylakoids. This
staining was, however, erratic and patchy, appearing in places where two membranes were in
contact. This differed from the finding of Uemura & Yoshida (1983) that thylakoid membranes
were prone to staining in sectioned intact material, but not in broken chloroplasts. The pellet
from the upper phase of a system with 50 mM-NaC1 consisted almost solely of small vesicles,
small sheets and possibly tubules (Fig. 3a). All of this material stained with PACP (Fig. 3c). The
material from the lower phase was much more heterogeneous, with vesicle diameters often
exceeding 1 Fm, and with vesicle groups suggesting unravelled thylakoids; granular material
was present (Fig. 3 4 . Staining of the lower phase with PACP was incomplete, only some of the
membranes reacting (Fig. 3f). In all of the bleached material Figs 2b, 3b and 3e), no
membranes remained stained (compare with Figs 2a, 3 a and 3 4 ; membranes electron dense in
PACP-treated sections (Figs 2c, 3c and 3f) are due solely to PACP-specific staining. Similar
results were obtained using material derived from P-deplete cells (as used in Fig. I), and from Preplete cells (as used in Fig. 4 and Tables 1 and 2). Thus electron microscopy indicated the
presence of plasma membrane and/or tonoplast in the upper phase, but did not distinguish
between the two. From the distribution of the 5’-NDase (Fig. I ) at least some of this material
must have been plasma membrane.
The enzyme marker usually used for plasma membrane is vanadate-sensitive ATPase, and for
the tonoplast, nitrate-sensitive ATPase and acid phosphatase (Thom & Komor, 1984; Mandala
& Taiz, 1985; Yoshida et al., 1986a, b). Because of the very high specific activity of 5’-NDase, it
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Diatom plasma membrane
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Fig. 3. Electron micrographs of the upper phase (a, b and c) and lower phase (d, e andf) of the twophase system described in the text. Uranyl acetate-lead citrate stained (a and d), periodic acid bleached
(b and e ) and PACP stained (c and fj sections. Bars 0.5 pm.
was not possible to measure the activity of ATPase or phosphatase, even at low pH, in a
preparation from P-deplete cells. In order to measure these enzymes P-replete cells were used in
which 5’-NDase activity cannot be detected (Flynn et al., 1986).
The activities of ATPase at pH 6.5 and of acid phosphatase (pH 5) in the upper phase
decreased, on addition of NaCl, in proportion to the partitioning of protein into the lower phase
(see Fig. 1). The specific activity of the acid phosphatase in the upper phase increased to 150%
in comparison with that of the microsomal fraction after the addition of 50 mM-NaC1; that of
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K. J . F L Y N N , H . OPIK A N D P. J . S Y R E T T
Table I . Ejects of inhibitors on ATPase activity at pH 6-5
--
The microsomal fraction of P-replete cells was partitioned in a two-phase system. The activity of
ATPase at pH 6.5 was measured as described in Methods, the total assay volume being 1 ml. All
inhibitors were added in 50 p1 of water except for NEM, DCCD and DES which were dissolved in
ethanol and 25 pl added together with 25 p1 water. Controls for the latter also contained a similar
amount of ethanol.
Upper phase
Inhibitor
(concn)
Lower phase
Specific
activity
[pmol Pi
(mg protein)-'
h-I]
Relative
activity
(percentage
of
control)
Specific
activity
[pmol Pi
(mg protein)-'
h-l]
Relative
activity
(percentage
of
control
Reference
5.8
2.9
4.9
100
49
84
8.8
4.0
11.2
100
45
127
Yoshida et af. (1986a)
Matsumoto & Yamaya
2-8
4.6
48
79
3.7
6.0
42
68
Serrano (1978)
Gallagher & Leonard
3.3
57
4.6
52
Sullivan & Volcani
4.0
5.8
68
100
4.4
7.8
50
88
5.7
98
7.3
82
Yoshida et al. (1983)
Gallagher & Leonard
(1 982)
Yoshida et af. (19863)
Control
Na3V04 (10 p ~ )
DES (100 VM)
CaC1, (5 mM)
NaN, (5 mM)
(1 984)
(1982)
(1 975)
Table 2. Ionic requirements of the upper phase ATPase at p H 6.5
The microsomal fraction of P-replete cells was partitioned in a two-phase system as described in the
text. The activity of ATPase, at pH 6.5 and 35 "C, was measured in the presence of various
concentrations of Na+, K+ and Mg2+with the osmotic potential maintained with sorbitol. In addition
to the ions added, 7.5 mM-Na+was present in all assays (derived from the NaATP and the NaCl present
in the two-phase system).
Ion
added
None
Na+
K+
Mg2+
Mg2+
K+
Mg2+
Na+
K+
Mg2+
N a+
K+
Mg2+
Na+
K+
Mg2+
Concn
(mM)
Relative
osmotic
potential
Specific activity
[pmol Pi
(rng protein)-' h-'1
200
200
2
100
100
100
100
0.4
2.4
3.2
3.9
4.2
60
4.8
100
100
4.7
97
100
3.9
80
3.9
80
200 t
8 J
100 7
100
;
Relative activity
9
50
81
87
ATPase decreased to 75 %. However, ATPases are very common throughout cellular
membranes and the crucial question is whether the ATPase partitioning into the upper phase
was the same as that in the lower.
Whilst the pH profile of the ATPase in the whole microsomal fraction, and of the ATPase in
the lower phase of a system with 50 mM-NaC1, showed a broad peak of activity between pH 6.5
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0.04
+
0.03
0.12
$
0.02
041
0.08
0.04
0
0
0
5
10
15
ZnClz concn (mM)
20
PH
Fig. 4. Effect of Zn2+ and H+ concentrations on the aggregation of vesicles partitioned from the
microsomal fraction of P-replete cells by a two-phase system. Samples from the upper ( 0 )and lower
(e)phases, of equal protein content (10 pg protein in 1 ml), were resuspended at various concentrations
of (a) ZnC1, and (b) H+ (i.e. pH). Apparent absorbance at 510 nm was measured against blanks of
0 mM-ZnC1, and pH 7.25 respectively.
and 8 - 5 , that of the ATPase in the upper phase showed a narrower peak of activity at about
pH 6.25-6.75. Further experiments using inhibitors (Table 1) showed that there were differences
between the ATPases isolated in the two phases when measured at pH 6.5. In particular, DES
inhibited ATPase in the upper phase, while stimulating activity in the lower; also K N 0 3 and
NaN3 had no effects on ATPase activity in the upper phase, but inhibited the lower phase
ATPase. Ca2+was more inhibitory towards lower phase ATPase. In comparison with the rate of
hydrolysis of ATP by enzymes in the upper phase, the rate of hydrolysis at pH 6.5 of AMP was
17%, and that for p-NPP (the acid phosphatase substrate) was 12%.
Experiments on the ionic requirements of the ATPase isolated in the upper phase indicated
the presence of a K+,Mg2+-stimulatedATPase (Table 2). The addition of K+ alone had a
greater effect than Na+, but Mg2+had a still greater effect. Highest activities were obtained in
the presence of both Mg2+and K+. Halving the osmotic potential (Table 2) had no effect on the
ATPase activity. Incubation of the upper phase vesicles with various concentrations of the nonionic detergent Triton X-100 did not increase the activity of the ATPase at pH 6.5, and
concentrations exceeding 3 % were inhibitory.
In preparations of identical protein content from each phase, the vesicles from the upper
phase aggregated less in response to high Zn2+or H+ concentrations than vesicles from the lower
phase (Fig. 4). This was particularly obvious in preparations treated with Zn2+ (Fig. 4a).
DISCUSSION
All the membranous material in the upper phase of a two phase system plus 50 mM-NaC1
stained with PACP (Fig. 3c). As, in intact cells, only the plasma membrane (and vesicles
between it and the cell wall - Flynn et al., 1986) and the tonoplast stained appreciably (Fig. 2c),
this therefore indicated that only these two membranes were present in the upper phase. The
distribution of 5’-NDase, a marker for plasma membrane (Gilkes & Weeks, 1977; Flynn et al.,
1986), and of acid phosphatase, a marker for tonoplast (Uemura & Yoshida, 1983; Yoshida et
al., 1983)and lysosomes (McMahon et al., 1977),was similar to those of membrane markers used
by Uemura & Yoshida (1983) and Yoshida et al. (1983). Thus, on addition of NaCl there was an
increasing partition of acid phosphatase (indicating tonoplast) into the lower phase, while the
percentage of 5’-NDase in the upper phase remained above 50%. Although the specific activity
of acid phosphatase in the upper phase did increase 1-5-fold(15-20% total activity remaining in
the upper phase), this compares poorly with the more than 4.5-fold increase in the specific
activity of 5‘-NDase ( 5 0 4 0 % total activity remaining in the upper phase). However, it should
be remembered that, because of the very high activity of the 5’-NDase, it was not possible to
assay both enzymes from the same cell preparation.
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K . J . F L Y N N , H . OPIK A N D P . J . S Y R E T T
The results from the measurements of the ATPase activities in the upper and lower phases
(pH optima; Fig. 4; Table 1) were consistent with enrichment of plasma membrane in the upper
phase, although undoubtedly some of the inhibitors would have been better used at different
concentrations. A proportion of plasma membrane partitioned into the lower phase (about 45 %
from the results of 5'-NDase measurements - Fig. l), so the activity of ATPase from the lower
phase at pH 6-5 (Table 1) would have been partly due to plasma membrane ATPase.
The effectsof the presence of Na+, K+, and Mg2+on the activity of the ATPase indicate that
it was a K+, Mg'+-stimulated ATPase. The original concentrations of these ions (200 mM-Na+,
200 mM-K+ and 8 mM-Mg2+)were chosen because these allowed maximum activity of the
plasma membrane ATPase of Nitzschia alba (Sullivan & Volcani, 1975). The residual activity
measured in the absence of these ions (except for the presence of 7.5 mM-Na+associated with the
substrate) was probably due to the presence of acid phosphatase in this fraction.
Because of the way in which the partition system functions, it may be expected that inside-out
plasma membrane vesicles would be partitioned into the lower phase together with the
endomembranes (Larsson et al., 1984). If this were so, then the activity of the ATPase of the
right-sided vesicles in the upper phase might be increased on addition of a detergent which
would allow a more rapid entry of ATP into the vesicles and hence increase activity (Larsson et
al., 1984; Wingstrand, 1985; Yoshida et al., 1986a). In P. tricornutum no increase in ATPase
activity was noted in preparations from the upper phase when the non-ionic detergent Triton X100 was added. However, as a significant proportion of the membranes present in the upper
phase were in small sheets, rather than closed vesicles, substrate diffusion to the site of
hydrolysis would not have been affected by addition of detergent.
The effects of cation concentration on the aggregation of vesicles from the upper and lower
phases were also consistent with the purification of plasma membrane and an absence of
endomembranes in the upper phase. Preparations of plasma membrane from SecaIe cereale (rye)
give a peak aggregation at pH 4 (Uemura & Yoshida, 1983). No such peak was seen in this
study, but the aggregation of membranes from the upper phase was considerably less than that of
membranes from the lower phase (Fig. 4b).
It is concluded that the two-polymer phase system gives a good separation of plasma
membrane from P.tricornutum. The principal contaminant, as judged by electron microscopy,
was tonoplast, and acid phosphatase assays indicated that this was probably less than 25 %of the
total membrane in this fraction.
The work described in this paper was funded by a grant from the SERC.
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