Plant Science, 75 (1991) 221--228
Elsevier Scientific Publishers Ireland Ltd.
221
Extracellular ferricyanide reduction and nitrate reductase activity
in the green alga Monoraphidium braunii
Alfonso
Corzo a, R e i n e r Plasa b a n d W o l f r a m R. Ullrich b
aDepartamento de Ecologia, Universidad de Mdtlaga, Campus Universitario de Teatinos, 29071 M{daga (Spain) and hlnstitut fiir
Botanik, Technische Hochschule, Schnittspahnstrasse 3, D-6100 Darmstadt (Germany)
(Received November 15th, 1990; revision received January 9th, 1991; accepted January 21st, 1991)
The unicellular green alga, Monoraphidium braunii, reduced extracellular ferricyanide at high rates. Impermeability of the membranes to ferri- and ferrocyanide was shown for untreated cells as for those permeabilized with 5% I-propanol. Fe starvation
stimulated reduction rates by 50%, light or glucose were ineffective. Extracellular acidification largely corresponded to formation of
the new anionic valence of ferrocyanide. 1-Propanol used as permeabilizer for NR assays in the same samples apparently inhibited
ferricyanide reduction, however addition of catalase showed that this was not due to interference with ferricyanide reduction but to
partial reoxidation by H20 2. Between ferricyanide reduction and nitrate reduction no competition was found in an 'in situ' system
with endogenous reductant in the presence of l-propanol, neither with 'induced' algae showing high NR activity nor with 'repressed'
algae grown in ammonium medium and showing only constitutive NR activity. According to this data the inducible high-activity NR
and the inducible nitrate uptake system are not responsible for reduction of ferricyanide. The plasmalemma-bound, low activity, NR
might have extracellular reducing activity, but also for this NR lack of competition in reduction of the two substrates indicated independent reactions in Monoraphidium.
Key words: ferricyanide; extracellular reduction; nitrate reductase; Monoraphidium braunii
Introduction
R e d u c t i o n o f ferricyanide by intact unicellular
algae has been r e p o r t e d by G o o d [1] a n d later by
N o v a k and M i k l a s h e v i c h [2] a n d N e g p f l r k o v ~ et
al. [3], r e d u c t i o n o f f e r r i o x a m i n e B a n d ferric
citrate m o r e recently by A l l n u t t a n d B o n n e r [4].
R e d u c i n g p o w e r for this r a p i d iron r e d u c t i o n has
been f o u n d to be derived f r o m p h o t o s y n t h e s i s ,
tricarboxylic or o x i d a t i v e p e n t o s e p h o s p h a t e
cycles. This extracellular r e d u c t i o n has been
studied with v a r i o u s electron a c c e p t o r s in higher
p l a n t s or algae, often with ferricyanide (hexac y a n o f e r r a t e III) as the electron a c c e p t o r [5--9].
Correspondence to: Alfonso Corzo, Departamento de Ecologia,
Universidad de M~ilaga, Campus Universitario de Teatinos,
29071 M~ilaga, Spain.
Abbreviations: Ferricyanide or ferrocyanide (Fe(CN)6, hexacyanoferrate 111 or ii), [Fe(CN)6]3- or [Fe(CN)6]4-; NR,
nitrate reductase.
A n essential question o f this process has been so
far, a n d still is, its p h y s i o l o g i c a l a n d e c o l o g i c a l
role. Jones et al. [10] for p h y t o p l a n k t o n , as o t h e r s
for roots, assume that it is m a i n l y used for
solubilizing a n d a b s o r b i n g trace elements, such as
iron a n d m a n g a n e s e , b u t this f u n c t i o n w o u l d n o t
require the high rates m e a s u r e d in s o m e p l a n t o r
algal materials.
Since Butz a n d J a c k s o n [11] p r o p o s e d a
p l a s m a l e m m a - b o u n d nitrate r e d u c t a s e being also
responsible for nitrate u p t a k e , the f u n c t i o n i n g o f
such an enzyme has been s u p p o r t e d a n d d o u b t e d
b y v a r i o u s e x p e r i m e n t a l d a t a [8,12--14]. Based on
i m m u n o l o g i c a l techniques, the idea has been exp e r i m e n t a l l y s u p p o r t e d . A c o n s t i t u t i v e p a r t o f the
nitrate reductase f o u n d in cells o f Chlorella a n d in
r o o t s has been s h o w n to be a t t a c h e d to the
plasmalemma, and addition of anti-nitratereductase a n t i b o d i e s c o u l d also inhibit nitrate
t r a n s p o r t , in isolated p r o t o p l a s t s as well as in
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
222
whole cells [15--17]. Reduction of siderophores
by nitrate reductase, but at much lower rates than
nitrate reduction, was demonstrated by other
groups [18]. With the same techniques but in the
diatom Thalassiosira, Jones and Morel [19] showed a triangular immunological correlation between
NR activity, nitrate uptake and extracellular ferricyanide reduction. NR antibody was detected to
(1) bind to the cell surface by using immunofluorescent labelling, (2) to inhibit the
plasmalemma redox activity in this organism and
(3) to inhibit primary amine production in the
presence of the impermeant electron acceptor
Cu2+-bathophenanthroline disulfonate. Jones and
Morel's hypothesis proposed according to these
data implies a direct competition for electrons
originating from NAD(P)H between nitrate reduction and extracellular ferricyanide reduction, both
being assumed to be activities of the same
plasmalemma redox chain.
The present paper reports on extracellular ferricyanide reduction activities of Monoraphidium
focusing mainly on the relationship between the
activities of nitrate reductase and the plasmalemma redox system. To enable measurements of ferricyanide reduction and NR activity in situ in the
same sample, a method was developed to eliminate
the interference of ferri- and ferrocyanide with
nitrite determination by reducing ferricyanide with
ascorbate and precipitating the formed ferrocyanide with Zn 2÷.
Material and Methods
Material
Monoraphidium braunii, strain 202-7d, from the
algal collection in G6ttingen was cultured synchronously with a 14/10-h light/dark rhythm at
29°C in a medium with the main nutrients: 8 mM
KNO3, 8 mM NaNO3, 0.1 mM Ca(NO3)2, 1 mM
MgSO4, 3.4 mM NaH2PO4, 0.8 mM NA2HPO 4,
zinc, iron, and trace elements, pH 6.2. For the experiments, cultures were taken from the exponential growth phase. Where indicated, the cells had
been cultured without iron and trace elements for
3 days.
N R assay in situ
A 1-ml sample of the algal suspension (cell
densities 2 0 ~ 0 #g chlorophyll ml-~ for NO3-grown, 60--90 #g chl ml-i for NH4+-grown cells)
was added to 1 ml of an assay mixture with 200
mM KNO3, 100 mM K-phosphate buffer (pH 7.5)
and 10% 1-propanol (i.e. double final concentrations). Five percent of 1-propanol were found to
be necessary for Monoraphidium. The samples
were incubated in the dark at 29°C. The reaction
was stopped at several points during the linear
reaction phase (0--20 min) by filtration through
membrane filters (cellulose acetate, 0.8 ~m pore
size, Sartorius, G6ttingen, F.R.G.). The generated
nitrite was determined immediately by photometry
of an azo dye [20].
This assay is called 'in situ' (not 'in vivo') in this
article in spite of some different use of these terms
[21], because the algae are used in a 'premortal'
rather than in a living state, while the enzyme is
assayed at its natural place within the cells (in
situ).
Ferricyanide reduction assay
A 1-ml sample of algal suspension in the culture
solution was added to 1 ml of a mixture containing
10 mM CaSO4, 30 mM Mes-Tris buffer (pH 7.5)
and 2 mM ferricyanide as K3Fe(CN)6. The
samples were incubated in the dark at 29°C for 15
min, and the reaction was stopped by filtration.
Disappearance of ferricyanide in the filtrate was
measured at 420 nm.
Ferricyanide and nitrate reduction in the same
samples
Prior to the experiment (30--60 rain) the algae
were filtered, resuspended in 5 mM CaSO4 and
kept in the standard culture conditions. The experiments were carried out with an algal cell density of 2 0 4 0 mg chl 1-1 (algae grown in nitrate)
and 60--90 mg chl 1-I (algae grown in ammonium) in a medium containing 100 mM KNO3,
50 mM phosphate buffer (pH 7.5), 2.5 mM CaSO4
and 5% (v/v) l-propanol. At the start of the experiments or after 10 rain, ferricyanide was added
to reach a final concentration of 1 raM. Control
samples were run without ferricyanide. All experiments were performed in the dark at 29°C.
Samples were removed at intervals, filtered
through membrane filters, and the remaining ferricyanide was measured at 420 rim. Both ferri-
223
,0
i
i
i
i
Extracellular acidification
i
0"
to
I.IJ
0.6 2 .,~~/
080.
0.4
,'@~,
0 10 20 30 40 50 60
,
,
,
,
It was determined by titration of the samples
with 25 mM KOH at the end of the experiments.
The original pH was 5.0 in these cases to avoid interference by HCO3-.
Chlorophyll
Total chlorophyll was determined with 1-ml
samples according to Senger [22] with hot methanol or, after extraction of the boiled algae, with
80% acetone.
NO~ (/aM)
Fig. i. Absorbance at 540 nm (E540) vs. K N O 2 concentration.
The colorimetric NO 2- measurement was performed in
samples of K N O z in distilled water ( a ) and in aliquots of the
same samples in which I m M ferricyanide had been added and
removed ( • ). n = 5, two independent analyses of each sample,
standard deviation not higher than symbol size.
Statistics
Figures and tables show either means of n experiments or single representative experiments out
of a group of replicates (n) as indicated.
Results
cyanide and ferrocyanide interfere with the
formation of the azo dye required for the determination of nitrite and, hence, had to be removed
prior to addition of the respective reagents. This
was achieved by adding 10/~1 of 0.1 M fresh ascorbic acid (to reduce ferricyanide to ferrocyanide)
and 50/~1 of 0.1 M ZnSO 4 (to precipitate the insoluble Zn2Fe(CN)6 ) to 1 ml of the sample. After
centrifugation at 4000 rev./min for 15 min nitrite
was measured in the supernatant. A comparison
between the efficiency of the original method of
nitrite determination and after addition and precipitation of ferrocyanide is shown in Fig. 1.
Monoraphidium braunii, similar to other algal
and higher plant cells, reduced ferricyanide at considerable rates (Table I). Starvation of iron for 3
days caused an increase in ferricyanide reduction
rates of more than 50%. Reduction was accompanied by an extracellular acidification that was a little stronger than accounted for by the generation
of the new anionic valence of ferrocyanide (H+/e = 1.16--1.17; Table I). Addition of glucose, which
was easily taken up by Monoraphidium and could
provide more reducing power [23], did not
stimulate ferricyanide reduction in the dark. In the
light, ferricyanide reduction was scarcely
Rates of ferricyanide reduction by Monoraphidium braunii in the dark under different conditions, pH 6.2. Rates in ~tmol
m g - l c h l h - t . Average rate (Fe-starved) 28.1 (n = 16), m a x i m u m 56.0, m i n i m u m 9.3. n, number of experiments. Values for comparison only from same experiments.
T a b l e 1.
Addition
[Fe(CN)6] 3- reduced
Fe-unstarved
Fe-starved
8.8 (2) a
13.8 (2) a
Fe-starved
+glucose
-glucose
K3Fe(CN) 6
Na3Fe(CN)6
44.7
46.5
22.1
20.2
(4)
(4)
(4)
(2)
Acidification
52.3 (7)
54.0 (4)
Ratios
-Fe/+Fe
1.57
H+/e H +/eK +/Na +
1.17
I. 16
1. I 0
aDirect comparison only; with indirect comparison, 25 experiments with unstarved, 16 with Fe-starved cells gave the same results.
224
stimulated (data not shown) but a rapid decomposition of ferrocyanide to free cyanide performed
by the algae (not by a cell-free system) was observed and led to the formation of blue Fe-complex
with the remaining ferricyanide.
As known from other plant material, the
plasmalemma is usually impermeable to ferricyanide so that its reduction must occur extracellularly. Since the in situ NR assay requires
the use of a membrane permeabilizer, it became
necessary to show that ferricyanide reduction was
also extracellular under these conditions, i.e. in the
presence of 5% 1-propanol, the most effective
permeabilizer in Monoraphidium (Nasa, unpublished results). Whole cells of Monoraphidium
were incubated in a solution containing 5 mM
CaSO 4, 50 mM phosphate buffer (pH 7.5), 5%
1-propanol and 1 mM ferricyanide for 15, 30, 45
and 60 min. The samples were then filtrated and
washed with the same, but ferricyanide-free, medium. The cells were deep-frozen with liquid N2,
disintegrated with a Microdismembrator (Braun,
Melsungen, F.R.G.), resuspended with distilled
water in an Ultra-Turrax and centrifuged at 4000
rev./min. The supernatant was used for assays of
both ferricyanide with Fe 2+, and ferrocyanide
with Fe 3+. Figure 2 shows that the total amount
of ferricyanide used was recovered in the cell-free
medium in the presence, in this case after reoxida-
~10,
I
I
I
I
°\
~6
z
o
4
o
0
u_
J
10
2'0
30
40
min
Fig. 2. Time course of ferricyanide reduction by Monoraphidium braunii in the presence ( [] ) and in the absence ( o ) of 5%
I-propanol. Closed symbols show concentration of ferricyanide
in the samples after reoxidation with H202 at the end of the
experiment.
' t-- 140
O
U
m-
cotolose
~nA
1
I=1+
w--
0
N ~o0
c"
O
u 60
~
~0
0
LL
control
Prop.( 5 % )
Fig. 3. Effect of catalase on the apparent ferricyanide reduction rate in the absence (control) and presence of 5%
l-propanol [Prop. (5%)]. Rates (p,mol [Fe(CN)6] 3- reduced
rag-l chl h-1) in % of control without catalase. Final concentration of catalase 650 units ml -I. Mean values, n = 3.
tion with H202, as well as in the absence of
1-propanol. This was confirmed with algal cells.
After their disruption, no formation of Feferricyanide complexes could be found with either
Fe 2+ or Fe 3+, even after several hours of in vivo
exposure to ferricyanide (n = 8).
In the presence of 1-propanol the rate of ferricyanide reduction appeared to be even to 40"/,,
lower than in its absence. Addition of catalase to
the medium completely abolished this 'inhibition'
(Fig. 3). Hence, 1-propanol apparently caused an
increase in formation of H202, which partly reoxidized the newly formed ferrocyanide and thus led
to an underestimation of the ferricyanide reduction rate in its presence. This result was confirmed
by kinetic analysis. In the absence of 1-propanol
the approximate kinetic parameters of external ferricyanide reduction in the dark were: Km -- 0.12
mM and Vma× ----26 ~tmol Fecy mg-I chl h-J. The
effect of l-propanol simulated non-competitive
inhibition: the apparent VmaX was much lower by
reoxidation of ferrocyanide with H202, but Km remained almost unchanged.
According to this evidence, ferricyanide reduction in the presence of 5% l-propanol (the
optimum
concentration
for
NR
assays
in Monoraphidium) proceeded at full rates.
225
Therefore, the experimental system used here was
adequate to reveal a possible competition between
extracellular ferricyanide reduction and internal or
membrane-bound nitrate reduction. However, the
addition of ferricyanide at three different concentrations to nitrate reducing algae (grown on
NO3- ) did not change the rate of NO2- production either in a shorter or longer period of time
(Figs. 4A and 4B). Both substrates were reduced
simultaneously without apparent interference, in
spite of the high rates (Fig. 4B).
According to the data from Chlorella and barley
roots, two different isoenzymes of N R have been
proposed, one cytoplasmic and inducible, the
other, with much lower activity, located in the
plasmalemma and constitutive, because it also
functions in Chlorella cells grown in NH4 ÷ media
[15,16]. In Monoraphidium this constitutive N R remained active when the cells had been grown with
5
0"3 1 A
r
'/
0.1
E
--
0
10 rain 20
0.41
~-
,
|
o. L
/7
,//
,,°
30
,
+ Fe (CN)6
13
I
z°
0
i
'
'
,
oot,-ol
,
10
rain 20
30
i
i,
Fig. 5. Time course of NO 2- f o r m a t i o n in NH4+-grown cells
with and without ferricyanide, in the presence of 5%
l-propanol (representative experiments, n = 4). (A) Control, no
[Fe(CN)6] 3- ( [] ), 1 m M [Fe(CN)6] 3- added after 10 min ( • ).
(B) Control, no [Fe(CN)6] 3- ( [] ), 1 m M [Fe(CN)6] 3- present
from the beginning ( • ).
'7
E 3
o
E
2
:3.
1:71
z
0
10
' rain 20'
3'o
15 ,5
7
o~
10 E
-6
E
5 -z
0
20 1.0 60 80 100 120
min
0 ~llJ
0
Fig. 4. Time course of N O 2- f o r m a t i o n in N O 3 - - g r o w n cells
with and without ferricyanide (added after 10 min, see arrows),
in the presence of 5% l - p r o p a n o l (representative experiments,
n = 4). (A) At different ferricyanide concentrations: no
[Fe(CN)6I 3- ( [] ), 0.1 m M ( • ), 0.5 m M ( • ), 1.0 m M ( * ). (B)
In c o m p a r i s o n with the time course of [Fe(CN)6] 3- reduction:
N O 2- no IFe(CN)6] 3- ( o ) ; N O 2 - , 1 m M [Fe(CN)6] 3- ( a ) ,
unreduced ferricyanide ( o ).
8 mM NH4C1 as the nitrogen source and
represented 5--8% of the total activity of
NO3--grown cells. Therefore, the true competition between ferricyanide and nitrate reduction at
the plasmalemma enzyme could have been concealed in the presence of cytoplasmic NR activity. However, as in nitrate-grown cells, the rate of NO 2formation in NHa+-grown cells did not show
any inhibition by ferricyanide, when added either
at the onset of the experiment or 10 min later
(Figs. 5A and 5B). In both cases, ferricyanide even
caused an increase in the NO2- formation rate, in
contrast to the data obtained with Thalassiosira
[191.
To exclude all kinds of competition also the inverse experiments were carried out. The rate of ferricyanide reduction in Monoraphidium braunii
was not altered, even slightly stimulated, by
KNO 3 up to 5 mM (Table II). A weak inhibition
226
Table II. Effect of KNO 3 on extracellular ferricyanide reduction in Monorapnidium braunii.
KNO3(mM )
0
0.1--5
10--100
>200
% activity
n
100
12
107
9
84
12
60
6
was detected between 10 and 50 mM, greater
nitrate inhibition only at very high K N O 3 concentrations beyond 100 mM, which means an
unspecific salt effect.
Discussion
Monoraphidium braunii shows rates of ferricyanide reduction that are in a similar order of
magnitude as with other plant material and algae
if calculated on an hourly and dry weight basis (for
Monoraphidium 1 mg chl = 24 mg dry wt.). With
Fe-starved algae the rates are even in the upper
range of the reported data [8,9] (10--56 tzmol
mg -1 chl h -l = 7--39 /xmol g-1 dry wt. min-I).
Glucose did not stimulate ferricyanide reduction,
even in the dark (Table I), although it is easily
taken up and can substantially stimulate nitrate
assimilation under some conditions [23]. The
simultaneous acidification in the medium can be
almost completely explained by the generation of
new anionic charges by the reduction from
(Fe(CN)6] 3- to [Fe(CN)6] 4- and charge equilibration by the weak cation, H + [24]. The small excess
in extracellular acidification is most easily explained by some uptake of K ÷, an ion available by the
use of 2 mM K3[Fe(CN)6].
The question of the physiological or ecological
role of extracellular reduction at such high rates
has not been clearly answered yet. The hypothesis
by Jones and Morel [19] assumes a role of the
plasmalemma redox chain in nitrate reduction,
nitrate uptake, and in reduction of other
substrates, while other authors propose a function
in stabilizing reduced membrane components and
protection against oxygen radicals [25].
While the bulk of nitrate reductase in higher
plants and algae has been found in the cytosol or
attached to inner membranes, part of the enzyme
has been localized in the plasmalemma in various
organisms as in Chlorella, Thalassiosira, maize and
barley roots. In Neurospora it was found to be attached to the tonoplast [26], in some unicellular
green algae within the chloroplasts and associated
to the pyrenoids [27].
The main evidence for the location in the
plasmalemma has come from immunological
studies, as has also the evidence for its function in
nitrate uptake at the plasmalemma [15--17]: NR
antibodies were able to inhibit nitrate uptake as
well as NR. In Thalassiosira they even inhibited
extracellular reduction of Cu2+-bathophenanthro line disulfonate.
In immunological localization some problems
might be involved, e.g. cross-reactions of
polyclonal NR-antibodies with different proteins.
This could be related to the complex structure of
NR which shares some of its prosthetic groups and
some partial activities with other enzymes.
NAD(P)H dehydrogenase activity and flavoprorein are common to various enzymes in the cell,
and even the molybdenum cofactor is very similar
to that of some oxidases [28].
Jones and Morel [19] proposed that the NR
located in the plasmalemma reduces intracellular
nitrate, extracellular electron acceptors and also
acts as a trans-plasmalemma proton pump. This
assumption implies competition for electrons from
NAD(P)H because both activities (reduction of
nitrate and of extracellular acceptors) must share
the initial part of the redox chain. Such a competition could only be prevented by supplying electrons to the terminal NR activity. Hence a mutual
inhibition as found for Cu 2+ bathophenanthroline
disulfonate in Thalassiosira was also expected for
ferricyanide and nitrate reduction in Monoraphidium. However, our results (Figs. 4 and 5)
do not show this competition, indicating that the
two activities are due to independent systems.
Moreover, NAD(P)H production rates and
NAD(P)H/NAD(P) ratios must be high enough to
completely satisfy both activities, at least under
our experimental conditions.
But there are also differences between the experiments with Thalassiosira and those reported
here, in the electron acceptor as well as in the assay
of nitrate reduction, for which Jones and Morel
227
used the production of primary amines and time
intervals of up to 24 h. Production of primary
amines includes the entire nitrate assimilation
pathway, i.e., in addition to nitrate reductase,
nitrite reductase and G O G A T need reducing
equivalents. In the experiments of the present
paper, nitrate reduction only was used to see the
competition
with
extracellular
ferricyanide
reduction.
Quite different redox activities have been
reported for the plasmalemma redox system [8,9].
The experiments of the present study use a redox
activity that accepts electrons from an intracellular
source and is stimulated by, but does not require,
Fe-starvation. According to the literature [8], the
plasmalemma redox system shows little specificity
with respect to electron acceptors: ferricyanide,
Fe3+-EDTA, cytochrome c, 02, ferrisiderophores,
dichlorophenol indophenol, Cu2÷-bathophen anthroline disulfonate etc., are easily reduced at
the plasmalemma.
According to Jones et al. [10] the redox system
in the plasmalemma of diatoms has a mid-point
redox potential between -100 mV and +94 mV.
Crane and Barr [8] reported that compounds with
a redox potential as low as 0 mV are able to serve
as electron acceptors at the membrane surface of
carrot cells. Since the redox potential of the couple
NO3-/NO2- is +400 mV [29], nitrate reduction
would be thermodynamically possible for this
unspecific system. However, in recent experiments
[30] a purified plasma membrane-associated electron transport protein from maize roots was able
to accept electrons from NAD(P)H to reduce ferricyanide and other acceptors but not at all nitrate.
It may be questioned if these properties may allow
for the low N R activity detected in plasmalemma
fractions of Chlorella and barley roots [16,31]. In
Monoraphidium 5--8% of total N R activity must
be accounted for. But even if it were so, addition
of nitrate should inhibit extracellular ferricyanide
reduction, or vice versa, by competition. The
results of this study show very little inhibition in
the presence of up to 50 mM K N O 3, greater
inhibition only at very high, unphysiological concentrations of NO 3- and K ÷. The increased
nitrate reducing activity of ammonium-grown
algae in the presence of ferricyanide can be ex-
plained by an increased supply of reducing
equivalents from the tricarboxylic acid or oxidative pentose phosphate cycles [24]. But apparently the electrons have an easier access to N R
than to the extracellular ferricyanide reduction
site.
From these data it can, at least, be excluded that
the bulk enzyme of N R is responsible for the extracellular reduction of ferric compounds at the
plasmalemma. It cannot be completely excluded
that the constitutive isoenzyme of NR as described
by Tischner [17] which may have a very low N R
activity compared with other reducing activities,
may have its main function in extracellular reduction, but no competition, rather independence,
could be observed in the present experiments. It is
not easy to imagine that the same protein should,
in addition, be responsible for nitrate transport, as
a low activity, constitutive transport system. If
there is a close relationship between constitutive
N R and constitutive nitrate transport, extracellular reduction seems to be independent of
this system in Monoraphidium.
Acknowledgements
The work was supported by a grant from the
Ministerio de Educaci6n y Ciencia in Madrid,
Spain (A.C.) and by the Deutsche Forschungsgemeinschaft, D F G (R.P. and W.R.U.). The authors
wish to thank Mrs. Ingrid Schroder for valuable
technical assistance.
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