Plant Physiol. (1981) 67, 212-215
0032-0889/81/67/0212/04/$00.50/0
Effects of Trypsin and Cations on Chloroplast Membranes
Received for publication April 11, 1980 and in revised form July 30, 1980
ROBERT C. JENNINGS, PAOLO D. GEROLA, FLAVIO M. GARLASCHI, AND GIORGIo FORTI
Centro di Studio del Consiglio Nazionale delle Ricerche per la Biologia Cellulare e Molecolare delle Piante,
Istituto di Scienze Botaniche, Via Giuseppe Colombo 60, 20133 Milan, Italy
ABSTRACT
MATERIALS AND METHODS
A mild typtic digestion of chloroplast membranes eliminates the effects
of saturatng concentrations of cations (3 to 5 milhimolar MgCI2) on
chlorophyll fluorescence yield, membrane stacking, and photosystem II
photochemical efficiency in spinach. At the same time, the negative surface
potential of the membranes i increased (by trypsin) as revealed by studies
with 9-aminoacridne. High concentrations of cations (25 to 100 millimolar
MgCI2) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations
added after trypsin treatment do not increase the photosystem II efficiency.
It is concluded that the "diffuse electrical layer" hypothesis of Barber et
aL (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily
explains the effect of trypsn in eminating the influence of saturating
concentratons of cations on chlwophyll fluorescence yield and membrane
stacking. However, the effect on photosystem II photochemical efficiency
seems to require another mechanism.
Chloroplasts were extracted from freshly harvested spinach
(Spinacia oleracea) leaves in a 30 mm Tricine buffer (pH 8.0) also
containing 10 mm NaCl and 0.4 M sucrose and, where indicated,
3 mM MgCl2. They were subsequently washed once and resuspended in the same buffer, unless otherwise stated. The reaction
solution was the same medium minus sucrose, unless otherwise
stated.
All fluorescence measurements were performed in a PerkinElmer MPF-3 spectrofluorimeter. 9-Aminoacridine fluorescence
was excited at 398 nm with an intensity of about 1000 ergs cm-2
s-i and measured at 456 nm. The chloroplast concentration was
20 ,ug Chl/ml, and the dye concentration was 25 ,UM. Under these
conditions, the addition of 10 mM MgCl2 increased the fluorescence yield, and no time-dependent quenching due to proton
pumping was observed at the low exciting light intensity employed.
Chl fluorescence was excited at 440 nm and measured at 683
nm at a Chl concentration of 4 isg/ml, unless otherwise stated.
Fluorescence induction was measured as previously described
(15). The actinic light intensity was 4000 ergs cm-2 s-' (Coming
4-96 filter) and the oscilloscope sweep time was 5 ms/division.
PSII activity was measured as 02 evolution in the presence of
the electron acceptor system 0.5 mM p-phenylendiamine 1 mM
potassium ferricyanide. Illumination was with white light of about
6000 ergs cm-2 s-1. Doubling the light intensity led to an 80 to
90%o increase in electron transport.
Fractionation of chloroplast membranes with digitonin was
performed essentially as described by Boardman et al. (6). Prior
to the fractionation step, the chloroplasts were washed with a
solution containing 30 mm Tricine (pH 7.4), 10 mm NaCl, and
also the appropriate concentration of MgCl2. Chloroplasts then
were suspended in the same medium at a Chl concentration of
300 pg/ml, and digitonin (2% w/v) was added to a final concentration of 0.5%. Treatment with digitonin was performed with
agitation at 0 C for 30 min when the chloroplast membranedetergent mixture was diluted with the same buffer, maintaining
unchanged the MgCl2 concentration. Separation of the heavy and
light membrane fractions was achieved by centrifugation at
lO,00Og for 30 min. Chl was extracted with 80%Yo acetone, and a/b
ratios were determined according to the equations of Mackinney
(18).
Electron microscopy was performed as previously described (8).
Trypsin (type I) and soybean trypsin inhibitor (type I-S) were
purchased from Sigma.
It has been known for many years that cations exert powerful
effects on chloroplast structure and electron transport activities.
Principal among these effects are cation-induced thylakoid membrane stacking (10, 12), increased PSII light utilization (21), increased PSII fluorescence yield (11, 21), and decreased PSI light
utilization (21). Attempts to understand the mechanism of these
effects have traditionally been made in terms of cation binding
sites (2, 7, 25). However, in 1977, an alternative possibility was
persuasively put forward by Barber et al. (4). They suggested that
it would be more fruitful to think in terms of diffuse electrical
layers at and very close to the thylakoid membrane surface to
explain many cation effects. Subsequent analysis demonstrated
the feasibility of this proposal (22-24), and the basic idea has
recently been modified in an attempt to take into account the
chemical diversity of different thylakoid regions (3).
Several years ago, we demonstrated that mild digestion of
thylakoid membranes with the proteolytic enzyme trypsin could
eliminate all of the above-mentioned cation effects (13, 14). Steinback et al. (25) subsequently verified these observations and
showed that the main effect of trypsin was to digest a small
fragment from the light-harvesting Chl a/b protein complex.
These data seem to support the idea that cation effects are
mediated by specific cation-binding sites, which should be located
on or near the light-harvesting complex. However, the possibility
that trypsin cleavage of the light-harvesting complex might lead
to an increase in the negative charge on the thylakoid membrane
surface by exposing previously hidden, negatively charged groups
remained uninvestigated.
Here, we investigated this possibility and attempted to distinguish between the "specific sites" and "diffuse electrical layer"
hypotheses for a number of cation effects.
RESULTS
Searle et al. (24) have demonstrated that use of 9-aminoacridine
as a probe for the electrostatic potential of the diffuse electrical
layer associated with chloroplast membranes. The idea is that the
monovalent cation, 9-aminoacridine, is attracted to the membrane
surface by the negative potential. Within the electrical double
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Plant Physiol. Vol. 67, 1981
213
TRYPSIN AND CATION EFFECTS ON THYLAKOIDS
layer, its fluorescence is quenched (probably by a mechanism of
concentration quenching). Cation addition, by decreasing the
negative surface potential, partially relieves this quenching. If
trypsin treatment of chloroplast membranes were to change the
surface-charge density and, hence, the negative potential of the
diffuse electrical layer, this should be reflected by changes in 9aminoacridine fluorescence.
Table I shows that mild digestion of chloroplast membranes
with trypsin in the presence or absence of Mg ions leads to a small,
but repeatable, decrease in 9-aminoacridine fluorescence. This
trypsin treatment was sufficient to halve the Chl fluorescence yield
in 4 min in the presence of 3 mM MgCl2 and to eliminate
completely the subsequent effect of 3 mM MgCl2 when trypsinization was performed in the absence of Mg ions. When trypsin
treatment was carried out in the presence of high Mg ion concentrations (50 mM), very small decreases in aminoacridine fluorescence were frequently observed which, however, were not statistically significant (unpublished data). It is not possible to quantitate experimentally increases of negative surface potential indicated by these fluorescence changes. However, on the basis of the
theoretical considerations of Barber and Searle (5) and assuming
that probe fluorescence responds similarly to equal changes in
surface potential induced by monovalent and divalent cations,
one can approximate that an increase in potential from -50 to
-60 mv would lead to a decrease in dye fluorescence of about 0.6
units under our experimental conditions. Electrostatic potential of
about -50 mv has been calculated for chloroplast membranes by
Barber et al. (4). Thus, we tentatively conclude that trypsin
treatment, sufficient to largely eliminate the stimulation of Chl
fluorescence by 3 mM MgCl2, is accompanied by an increase of
approximately 20%o of the negative surface potential. This value is
intended to be understood as being orientative and indicative of
the probable order of magnitude of the potential changes observed.
These data do not, however, demonstrate that such changes are
involved in the mechanism by which trypsin suppresses cation
effects on chloroplast membranes.
The diffuse electrical layer theory would suggest that trypsin
may counteract the effects of saturating concentrations of cations,
increasing the negative membrane surface potential. In this circumstance, in order to reduce the electric potential to values
previously attained by relatively low cation concentration, higher
concentrations would be needed. We have titrated a number of
cation-sensitive phenomena, which are influenced by trypsin
digestion, before and after proteolytic treatment. These are: (a)
cation-induced Chl fluorescence yield, (b) cation-induced memTable I. Effect of Trypsin Digestion of Chloroplast Membranes on
Fluorescence Yield of 9-Aminoacridine
Chloroplasts had been prepared with 3 mm MgCl2 in all buffers.
Washing and resuspension of the chloroplasts were performed with a
medium containing 15 mM Tricine (pH 8.0), 5 m. NaCl, 3 mN MgC12,
and 0.4 M sucrose; trypsinization and fluorescence measurements were
performed in the same medium minus sucrose in the presence or absence
of 3 mm MgCl2. Before adding trypsin (I ug/ml), the chloroplasts were
incubated at 20 ug Chl/ml for 3 to 4 min in the reaction buffer, containing
25 Am 9-aminoacridine. Data are arbitrary units of fluorescence.
Fluorescence Yield after Time with Trypsin'
Additions
2 min
4 min
6 min
0 min
23.9
23.5
23.1
23.0
None
30.8
31.0
30.3
30.2
MgCI2
of
control
and
as
a
function
of time
*Variabilty
trypsin-treated samples
was estimated by linear regression. The difference between the regression
coefficients of the two groups (control, + trypsin), assessed by analysis of
variance (parallelism test), was significant at the 99% confidence level.
Control values did not change during the experiment.
brane stacking, and (c) cation-induced increase in PSII photochemical efficiency.
Figure 1 represents an experiment in which the Chl fluorescence
yield was titrated with different MgCl2 concentrations before and
after a brief treatment with trypsin. In the absence of proteolytic
digestion, the concentration of MgCl2 required to elicit half the
maximal fluorescence response was around 0.6 mm and, after
trypsin treatment, about 10 mM MgCl2 was required. However,
even at the highest Mg ion concentration, the reversal of the
trypsin effect was not complete. It seemed possible that this may
have been associated with the substantial decrease of fluorescence
yield caused by trypsin in the absence of Mg ions. This suggests
that trypsin treatment lowers the "basal" fluorescence yield (not
due to added cations), as do experiments in which pronounced
trypsin effects have been measured also in the presence of EDTA
(unpublished data). Thus, if the data are replotted as the ratio of
this basal fluorescence (Fig. 1B), the reversal of trypsin effects by
high Mg ion concentrations is almost complete.
It is well known that cations increase Chl fluorescence largely
by increasing the variable fluorescence (21). To check if the same
applies upon addition of high Mg ion concentrations after trypsin
digestion, we performed fluorescence induction studies. They show
that high Mg2e concentrations after trypsin treatment increase
almost exclusively the variable fluorescence (Table II).
Figure 2 shows electron micrograph pictures of chloroplasts
subjected to trypsinization sufficient to eliminate almost all membrane stacking, in the presence of 3 mM MgCl2. Subsequent
addition of high concentrations of MgCl2 caused the reappearance
of membrane appression. Although this stacking is not morphologically identical to that of chloroplasts not treated with trypsin,
where clearly distinguishable grana can be seen, it frequently
occurs in distinct zones in which a number of membranes seem to
adhere together. To clarify whether or not these structures can be
considered to be grana, we performed digitonin fractionation
studies (Table III). Goodchild and Park (9) have shown that the
A
C.)a)
75-
C.)(L)
Co
U)
0
50-i
r'l
^
-"101-...
c)
25-
B
.2-
2)
Cc)
1. 82. (
a)
c00
o
L
X
'
1.1,oH
%,I --q
I
I
10
20
I
I
30 40
I
50
80
Mg C12 ( mM)
FIG. 1. Titration of the Chl fluorescence yield with MgCl2 before and
after treatment of the thylakoid membranes with trypsin. Chloroplasts
preparation and tryptic digestion were performed in the absence of MgC12.
Proteolytic digestion was terminated by addition of a 50-fold excess of
trypsin inhibitor, at which time the different concentrations of Mg2+ ions
were added. The fluorescence yield was measured 4 min later. A, the
unmodified fluorescence yields; B, the data from the same experiment are
plotted as the ratio of fluorescence in the presence of Mg2e to fluorescence
measured in the absence of MgCl2. (0), minus trypsin; (@), plus trypsin
(2 pg/mL 1 min). The Chl concentration was 20 Ag/ml.
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. _§ *: ~ t . ,
JENNINGS ET AL.
214
Table IL Effect of Dfferent Concentrations of MgClk on Variabk and
Nonvaruabk Components of Ch Fluorescence, before and after Treatment
with Trypsin
Chloroplasts were prepared in the presence of 3 mm MgCl2 and then
treated (at 20 ug Chl/ml) with or without trypsin (Tryp; I ,g/ml, 4 min)
in the presence of 3 mM MgC12. Trypsin digestion was terminated upon
addition of a 50-fold excess of trypsin inhibitor. Chloroplasts were subsequently washed in a solution containing 15 mM Tricine (pH 8.0), 5 mM
NaCI, and 0.4 M sucrose and resuspended in the same buffer at different
concentrations of MgCl2. Measurements of fluorescence induction were
carried out as described. Data are arbitrary units of fluorescence.
Fluorescence Induction at Following Magnesium
Concentrations
Fluorescence
5 mM
0
Parameter
50 mM
Variable
Nonvariable
-Tryp +Tryp
17
34
17
21
p~~~W7
w
-Tryp +Tryp -Tryp +Tryp
20
103
39
111
20
38
21
28
*5
t g
ass 4
w
wS
*_
'ssW
* O>;
,.
f
f\
I
784_
<
I
Plant Physiol. Vol. 67, 1981
Table III. Effect of Various Concentrations of Mg2+ IonS on
Fractionation of Chloroplast Membranes by Digitonin, before and after
Treatment of Membranes with Trypsin
For details of the experimental procedures, see Figure 2 and "Materials
and Methods." Trypsin treatment was 1.2 p/ml for 4.5 min. Ratio
indicates the Chl a/b ratio and Yield indicates the percentage of the total
Chl a + Chl b found in that particular fraction.
-Trypsin
+Trypsin
Mg2+
Concn.
Light Fraction
Heavy Fraction
Light Fraction
Heavy Fraction
Ratio Yield Ratio Yield Ratio Yield Ratio Yield
0
5
50
100
2.83
4.03
3.93
3.66
61
15
19
30
%
%
%
mM
2.68
2.63
2.53
2.42
39
85
81
70
2.72
2.52
3.17
3.64
83
49
33
40
%
2.66
2.93
2.52
2.13
17
51
67
60
Table IV. Effect of various Concentrations of MgCl2 on Reduction ofpPhenylenediamine at Low Light Intensity, before and after Treatment with
Trypsin
Trypsin treatment (70 s, 1.6 pg/ml) of chloroplasts (20 pug Chl/ml) was
terminated by addition of a 50-fold excess of inhibitor. Units are nano
electron equivalents/pg Chl.h. Incubation with MgCl2 was for 4 min
before commencing the measurement.
Effect of Following Salt Concentrations:
Treatment
0
5 mM 10 mM 15 mM 25 mM 50 mM
72
48
68
70
61
61
-Trypsin
+ Trypsin
46
48
52
46
42
50
MgCl2 concentrations after trypsin treatment are in fact grana.
Table III shows that, although there is a clear relationship between
the Mg2e-induced changes in Chl a/b ratio and yield in the light
and heavy digitonin fractions in the absence of trypsin treatment,
this is not the case after digestion with trypsin. In the latter case,
Mg2+ exerts a strong influence on the yield of the light and heavy
fractions while having no effect on the Chl a/b ratios. This aspect
. =
~~~~~~~~f.,
is treated in more detail elsewhere (17).
In Table IV, data are presented from an experiment in which
the effect of Mg ions on PSII light utilization was determined at
FIG. 2. The effect of high concentrations of MgCl2 on chloroplast different MgCl2 concentrations, with chloroplasts treated or not
structure after tryptic digestion. Chloroplasts were prepared with 3 mm with trypsin. This treatment was sufficient to eliminate the wellMgC12. Trypsin (1 pg/ml) treatment was performed in the presence of 3 known effect of Mg2+ on PSII light utilization. Increasing the
concentration of MgCl2 up to 50 mm after trypsin treatment did
mM MgCl2, at a Chl concentration of 20 ug/ml. The reaction was terminated after 3 min by addition of a 50-fold excess of trypsin inhibitor and, not reverse this effect of trypsin to the slightest degree.
3 min later, MgCl2 was added as indicated. Control chloroplasts were
suspended under the same conditions but without Mg2e ions, for 6 min
before MgCl2 additions. Control and treated chloroplasts then were incubated in an ice bath for 30 min and fixed for electron microscopy. a,
control, 3 mM MgC12; b, control, 50 mM MgC12; c, control, 100 mM MgC12;
d, trypsin, 3 mM MgCl2; e, trypsin, 50 mM MgCl2; f, trypsin, 100 mm
MgCl2. Bars, 1 pm.
DISCUSSION
Experiments reported here with 9-aminoacridine demonstrate
that trypsin treatment of chloroplast membranes significantly
increases the negative electrostatic potential at and near the membrane surface. The proteolytic treatment required to elicit this
effect is approximately equivalent to that required to eliminate the
light digitonin fraction is enriched in stroma lamellae and the effects of saturating concentrations of Mg2 ions on Chl fluoresheavy fraction is enriched in grana lamellae. The data show that, cence yield, membrane stacking, and PSII light utilization. Inin the absence of trypsin treatment, MgCl2 at a concentration of creasing the cation concentration after trypsin digestion is shown
5 mm was sufficient to cause the well-documented enrichment in
to be effective in substantially reversing these trypsin effects on
Chl a of the light membrane fraction. Higher salt concentrations both the Chl fluorescence yield and membrane stackg. This
resulted in a slight decrease in this parameter. After trypsin phenomenon is predicted by the diffuse electrical layer hypothesis
treatment, however, 5 mm MgCl2 was ineffective in causing the (3, 4), where cations are expected to lower the negative surface
enrichment of the light digitonin fraction in Chl a. This was potential by concentrating in a diffuse layer near the membrane
achieved upon increasing the MgCl2 to 50 and 100 mm. We surface. Thus, any increase in negative surface potential should be
conclude that the membrane appression structures seen at high diminished by increasing the cation concentration in the medium.
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Plant Physiol. Vol. 67, 1981
TRYPSIN AND CATION EFFECTS ON THYLAKOIDS
We conclude that the diffuse electrical layer hypothesis satisfactorily explains the effects of trypsin in suppressing the effects of
saturating concentrations of cations on Chl fluorescence yield and
membrane stacking. Previous studies (1, 10, 14) have suggested
that the cation-stimulated Chl fluorescence is associated with
grana formation. The data presented here, which demonstrate a
similar basic mechanism for both phenomena, support this conclusion.
Increasing the cation concentration after trypsin digestion does
not increase PSII efficiency. Thus, it is clear that the trypsin
suppression of this cation effect is not via the increase in electrostatic potential. It probably indicates that the basic cation interaction required to stimulate PSII is different from that described
by the diffuse electrical layer hypothesis. It is possible that specific
cation binding sites are involved. Such a conclusion is in complete
accord with our earlier data, indicating that the effect of cations
on PSII is able to be dissociated from the other effects on fluorescence and membrane stacking (13, 14, 16).
Recently both Mullet and Arntzen (20) and McDonnel and
Staehlin (19) have demonstrated that, when the light-harvesting
Chl-protein complex is incorporated into artificial membranes,
cations can cause adhesion between these membrane surfaces.
This "membrane stacking" is eliminated by trypsin treatment.
These data are interpreted as demonstrating that this Chl-protein
complex is the "stacking factor." It seems likely that the trypsin
effects reported by these authors (19, 20) can be explained in
terms of an increase in the negative surface potential of the
liposome-Chl-protein complex membranes and that the cationmediated stacking is controlled by the dffuse electrical layer
associated with these membranes. The data of Mullet and Arntzen
(20) and McDonnel and Staehlin (19) may demonstrate a molecular specificity at the level of the weak attractive forces required
to hold the membranes together once the negative electrical field
has been sufficiently masked by cations. However, this has not yet
been definitively demonstrated and would require that other
thylakoid membrane proteins when incorporated into artificial
membranes do not mediate cation-induced stacking.
ADDENDUM
After this work had been submitted for publication, an article
by Nakatani and Barber (Nakatani HY and Barber J 1980
Biochim Biophys Acta 591: 82-91) was published, in which they
demonstrate, using the technique of particle electrophoresis, that
trypsin treatment of chloroplast membranes probably increases
the negative surface potential.
LITERATURE CITED
1. ARGYROUDI-AKOYUNOGLOU JH, G AKOYUNOGLOU 1977 Correlation between
cation-induced formation ofheavy subchloroplast fractions and cation-induced
increase in chlorophyll a fluorescence yield in Tricine-washed chloroplasts.
215
Arch Biochem Biophys 179: 370-377
2. ARNTZN Ca, CL Drrro 1976 Effects of cations upon chloroplast membrane
subunit interaction and excitation energy distribution. Biochim Biophys Acta
449: 259-274
3. BmBER J, WS CHOW 1979 A mechanism for controlling the stacking and
unstacking of chloroplast thylakoid membranes. FEBS Lett 105: 5-10
4. BARBER J, J MiLLs, A LovE 1977 Electrical-diffuse layers and their influence on
photosynthetic processes. FEBS Lett 74: 174-181
5. BARER J, GFW SEARn! 1979 Double-layer theory and the effect of pH on
cation-induced chlorophyll fluorescence. FEBS Lett 103: 241-245
6. BOARDMAN NK, SW THORNE, JM ANDERSON 1966 Fluorescence properties of
particles obtaied by digitonin fragmentation of spinach chloroplasts. Proc
Natl Acad Sci USA 56: 586-593
7. DAvis DJ, EL GROSS 1976 Protein-protein interactions of the light-harvesting
chlorophyll a/b protein. II. Evidence for two stages of cation-independent
association. Biochim Biophys Acta 449: 554-564
8. GEtoLA PD, RC JENNINGs, G FORTI, FM GARLASCHI 1979 Influence of protons
on thylakoid membrane stacking. Plant Sci Lett 16: 249-254
9. GooDCHnm DJ, RB PARK 1971 Further evidence for stroma lamellae as a source
of photosystem I fractions from spinach chloroplasts. Biochim Biophys Acta
226: 393-399
10. GROSS EL, SH PRASmtx 1974 Correlation between monovalent cation-induced
decreases in chlorophyll a fluorescence and chloroplast structural changes.
Arch Biochem Biophys 164: 460-468
11. HomANN P 1969 Cation effects on the fluorescence of isolated chloroplasts. Plant
Physiol 44: 932-936
12. IZAWA S, NE GOOD 1966 Effect of salts and electron transport on the conformation of isolated chloroplasts. II. Electron microscopy. Plant Physiol 41: 544552
13. JENNINGS RC, G FORTI 1978 Interactions of cations with thylakoid membranes.
In GF Azzone, M Avron, JC Metcalfe, E Quagliariello, N Siliprandi, eds, The
Proton and Calcium Pumps. Elsevier/North-Holland, Amsterdam, pp 93-104
14. JENNINGS RC, G FORTI, PD GEROLA, FM GARLASCHI 1978 Studies on cationinduced thylakoid membrane stacking, fluorescence yield, and photochemical
efficiency. Plant Physiol 62: 879-884
15. JENNINGS RC, FM GARLAscm, G FORTI 1976 Studies on the slow fluorescence
decline in isolated chloroplasts. Biochim Biophys Acta 423: 264-274
16. JENNINGS RC, FM GACIu.cH, G FORTI, PD GEROLA 1978 Cations and energy
spillover in chloroplasts. Plant Sci Lett 13: 1-8
17. JENNINGS RC, PD GEROLA, FM GARLAscHI, G FORTI 1980 Studies on the
fractionation by digitonin of chloroplast membranes. FEBS Lett II 5: 39-42
18. MAcIUNNEY G 1941 Absorption of light by chlorophyll solutions. J Biol Chem
140: 315-322
19. McDoNNEL A, LA STAEHUN 1980 Adhesion between liposomes mediated by the
chlorophyll a/b light harvesting complex isolated from chloroplast membranes.
J Cell Biol 84: 40-56
20. MuLLET JE, Ca ARNzrEN 1980 Stimulation of grana stacking in a model
membrane system. Mediation by a purified light-harvesting pigment-protein
complex from chloroplasts. Biochim Biophys Acta 589: 100-117
21. MURATA N 1969 Magnesium ion-dependent distribution of excitation energy
between two pigment systems in spinach chloroplasts. Biochim Biophys Acta
189: 171-181
22. NAKTANI HY, J BmBER, JA FORRESTER 1978 Surface charges on chloroplast
membranes as studied by particle electrophoresis. Biochim Biophys Acta 504:
215-225
23. NAKATANI HY, J BARBER, MJ MINSIU 1979 The influence of the thylakoid
membrane surface properties on the distribution of ions in chloroplasts.
Biochim Biophys Acta 545: 24-35
24. SEARLE GFW, J BARBER, JD MILLS 1977 9-Aminoacridine as a probe of the
electrical double layer associated with chloroplast thylakoid membranes.
Biochim Biophys Acta 461: 413-425
25. STEINBACK KE, JJ BURKYE, CJ ARNTzEN 1979 Evidence for the role of surface
exposed segments of the light-harvesting complex in cation-mediated control
of chloroplast structure and function. Arch Biochem Biophys 195: 546-557
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