Download The rhythm of protein synthesis does not depend on oscillations of

Journal of Cell Science 103, 363-370 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
363
The rhythm of protein synthesis does not depend on oscillations of ATP
level
VSEVOLOD Y. BRODSKY1-*, PETR Y. BOIKOV2, NATALIA V. NECHAEVA1, YURI G.
YUROVITSKY1, TATYANA E. NOVIKOVA1, VALENTINA I. FATEEVA1 and NATALIA A.
SHEVCHENKO2
1
2
Department of Cytology, Institute of Developmental Biology, 26 Vavilov St, 117808 Moscow, Russia
Department of Molecular Biology, Institute of Chemical Physics, Moscow, Chernogolovka, Russia
•Author for correspondence
Summary
A rhythm of the [3H]leucine incorporation rate with a
period of about one hour (circahoralian rhythm) has
been found in rat hepatocytes grown in vitro as a
monolayer and in liver organ culture. The periodicity of
the incorporation rate remained after correction for
changes in leucine pool size. A similar periodicity of the
leucine incorporation rate was detected in a cell-free
system prepared from rat hepatocytes. We have also
found circahoralian oscillations of the ATP level and
similar oscillations of the leucine tRNA aminoacvlation
rate in a hepatocyte monolayer. The addition of 1 mM
ADP to the culture resulted in a considerable increase in
the ATP level in the cells, but the rhythm of protein
synthesis was retained under these conditions. The
conclusion that there is a flexible association between
changes in the ATP and GTP levels on the one hand, and
oscillations of the protein synthesis rate on the other, is
also supported by experiments with a cell-free system, in
which the rhythm of protein synthesis rate was observed
in the presence of excess ATP and GTP. We propose an
hypothesis to explain the fractal pattern of circahoralian
metabolic rhythms.
Introduction
Jasumasu, 1976) and a similar rhythm was described for
the activity of ornithine decarboxylase, a key enzyme of
polyamine synthesis, in hepatocytes in vitro (Jarigin et
a!., 1978). In these two systems a rhythm of cyclic AMP
levels has also been described (Jarigin et al., 1979;
Ishida and Jasumasu, 1982). Oscillations of total
adenylic nucleotide content have been described in the
rat muscle (Thornheim and Loewenstein, 1973). An
important result was the detection of circahoralian
periodicity of ATP, ADP and AMP levels, in association with a similar periodicity in the respiration and
protein content of cells in a synchronized culture of
Acanthamoeba (Lloyd et al., 1982).
In the present study we have examined the ATP
level, tRNA aminoacylation and the incorporation of
leucine into proteins of hepatocytes grown in vitro.
Earlier (Jarigin et al., 1978, 1979) we found circahoralian rhythms of cAMP and ornithine decarboxylase
activity in such cultures. Now we have also studied
changes in the rate of protein synthesis in a cell-free
system from rat hepatocytes.
The first observations of periodic oscillations in the
protein content of cells were made in the 1950s
(Brodsky, 1959). Later it was discovered that the rate of
amino acid incorporation into proteins had a similar
periodicity of almost an hour (Kalendo and Kusin,
1966; Mano, 1970). Approximately at the same time, a
similar periodicity was found in the activity of various
enzymes (see, for example, Masters and Donachie,
1966; Gilbert, 1974). All these rhythms have been
named circahoralian to differentiate them from circadian and also ultradian rhythms (Brodsky, 1975). To
date the literature on circahoralian rhythms of protein
metabolism contains at least 100 titles (for references
see Brodsky and Nechaeva, 1988).
There has been considerable progress in the detection of circahoralian rhythms of physiologically active
metabolites that may affect the rate of protein synthesis. A rhythm of polyamine concentrations was
observed in cleaving sea urchin embryos (Kusunoki and
Key words: hepatocytes in vitro, cell metabolic rhythms,
protein synthesis, ATP, cell-free system.
364
V. Y. Brodsky and others
Materials and methods
Cultivation of hepatocytes
The technique of Berry and Friend (1969) was used with some
modifications. Rat liver was perfused with calcium-free
Hanks' solution containing 0.5 mM EDTAfor 10 min at 37°C.
This was followed by perfusion with 0.5% collagenase in
Eagle's medium. The liver was isolated, freed from the
capsule, placed in a flask with Eagle's medium and carefully
stirred for 2-3 min. The resulting cell suspension was passed
three times through a nylon filter (pore diameter 70 ^m), with
centrifugation each time at 200 revs/min for 5 min. The final
suspension contained about 95% of intact hepatocytes. A 3 ml
sample of the suspension containing approximately 3X106
hepatocytes was placed in a Petri dish, 60 mm in diameter,
with collagen-coated coverslips. The incubation medium
contained: Eagle's medium, 80%; foetal calf serum, 20%;
penicillin and streptomycin, 100 /ig/ml each. The gas phase
contained 5% carbon dioxide and 95% air. The medium was
replaced after 1-2 h and then after 2, 4 and 48 h of incubation.
The actual experiments were performed on day 3 after plating
the cells. The details of organ cultivation have been described
(Nechaeva et al., 1970).
Studies of protein synthesis
Every 10 min a coverslip with a cell monolayer was taken from
the dish with medium and incubated for 10 min with tritiated
leucine (25 ^Ci/ml, specific activity 75 Ci/mmol). After 10 min
of incubation the cells were washed several times with cold
(4°C) medium containing an excess of unlabeled leucine and
treated with cold 5% perchloric acid for 90 min. Then the
monolayer was washed with ethyl alcohol and the proteins
were dissolved in hyamine. Scintillation cocktail was added
and protein radioactivity measured using an SL-30 (Intertechnique) scintillation counter. The radioactivity in the acidsoluble fraction (extracted with perchloric acid) was measured
in a similar way. The incorporation was calculated per
milligram of protein or expressed with respect to the total
radioactivity of the acid-soluble fraction plus proteins.
Changes in ATP level
Perchloric acid extracts obtained as described above after
neutralization were also used to measure the ATP level. This
was done in a reaction mixture containing 5.05 mM 3phosphoglyceric acid, 3.33 mM magnesium sulfate, 0.9 mM
EDTA and an excess of phosphoglycerate kinase and
glyceraldehyde phosphate dehydrogenase (Jaworen and
Gruber, 1970). The measurements were performed using an
SF-4 spectrophotometer. The ATP values were normalized
for DNA content in the cell sample.
Measurement of aminoacylation
After 10 min of incubation with labeled leucine the cells were
washed with medium containing an excess of unlabeled
leucine and 0.01 M pyrophosphate, which is an inhibitor of
tRNA aminoacylation. Thereafter, the monolayer was
quickly treated with cold (4°C) trichloroacetic acid and frozen
at —70°C. In order to determine the amount of [3H]leucine
radioactivity in aminoacyl tRNA (aa-tRNA) the cells were
washed three times with ice-cold 5% trichloroacetic acid
(TCA) and centrifuged at 1000 g for 10 min at 4°C. TCA (5%)
was added to the washed precipitate, the material was heated
to 90°C and incubated at this temperature for 15 min for aatRNA hydrolysis. The mixture was chilled and after centrifugation the radioactivity in the supernatant was measured. This
activity was assumed to represent the level of leucyl-tRNA.
The precipitate was washed with cold TCA, dissolved in 1 M
NaOH, diluted with water to a concentration of 0.1 M NaOH
and the protein concentration and radioactivity were determined.
Preparation of a cell-free system and analysis of
protein synthesis
A cell-free system was prepared from rat liver as described by
Eisenstein and Harper (1984). This method was also used for
the study of protein synthesis in a cell-free system. The
postmitochondrial supernatant (4 mg protein/ml) was incubated with 125 mM potassium acetate, 2 mM magnesium
acetate, 50 mM HEPES (pH 7.55), 2.5 mM dithiothreitol, 20
mM sodium phosphocreatine, 250 jig/ml creatine kinase, 2
mM ATP, 1 mM GTP, and 200 ftM of each amino acid,
including leucine labeled with tritium or radioactive carbon.
The temperature was kept at 30°C. Samples of the reaction
mixture were taken every 10 min for 2 hours (their volume
was 10, 50 or 100 /il in different experiments yielding similar
results). They were applied to pieces of filter paper and air
dried. Filters were washed with cold 10% TCA containing an
excess of non-radioactive leucine, treated with boiling TCA,
washed again with cold 5% TCA, dried, and then the
radioactivity was measured with a SL-30 scintillation counter.
Results
Pulse labeling of cell proteins in the hepatocyte
monolayer detected some periodicity in the leucine
incorporation rate (Fig. 1). Three different investigators studied the given culture over a certain period of
time. The three resulting curves have similar shapes.
The integral curve (showing errors for three specimens
for each time point) shows oscillations in leucine
incorporation. The incorporation for each period
increases gradually and then falls steeply. Examples of a
different kinetics with a gradual decrease in incorporation during the periods are shown in Figs 2 and 3.
In order to estimate the exact kinetics, the samples
were taken at short intervals. Fig. 2 shows a curve for
the samples taken at 5 min intervals, as well as a 10 min
transformation of the curve. In the latter curve the first
point coincides with the second point of the 5 min
curve, and then every second point was taken. Samples
taken at both 5 min and 10 min intervals showed similar
kinetics of leucine incorporation.
The incorporation rate (I) was normalized for the size
of the free leucine pool (P) by using either the l/P ratio
or the total radioactivity value (I+P=T). In the latter
case, one of the T values was taken as 1.0; other values
expressed as its parts (Ti/T,) represent the correction
factor for /*. Both curves obtained after such corrections showed similar incorporation kinetics, reflecting
similar protein synthesis rates (Fig. 3).
In addition to corrections for the labeled pool the use
of relative values normalizes specimens in terms of
weight and cell number. When studying cell monolayers
in vitro (every specimen is on a separate coverslip) it is
impossible to determine the exact number of cells in
each specimen. Therefore, the absolute values of the
incorporation rate and pool size in different specimens
cannot be compared. When using LjPx or another way
ATP and protein synthesis rhythms
120
0
Time(min)
80
365
120
0
Fig. 1. Kinetics of [3H]leucine incorporation into
hepatocyte proteins in liver slices in organ culture. Here
and in other experiments (except that shown in Fig. 2)
individual samples of the same culture were incubated one
by one with labeled leucine for 10 min. Three samples
were taken for each time point; consequently, three curves
were obtained (•, *, • ) . Mean values with standard errors
are shown on the averaged curve (bottom right). This and
other figures were made using an IBM PC/AT computer
with Supercalc software for the calculations and Harvard
Graphics software for plotting the results.
of expressing the relative incorporation, the cell
number, which is identical in the numerator and
denominator, is cancelled. In the Discussion we present
results of special experiments on the incorporation
kinetics and pool size as well as questions related to the
endogenous pool.
In different experiments, oscillation periods, even for
the same culture, varied from 30 to 80 min (see also
other figures). Similar oscillation patterns were observed in hepatocyte monolayers in vitro (Figs 3 and 8),
in organ culture (Figs 1 and 2), and in cells in vivo (see
Discussion). We define such oscillations as circahoralian.
Oscillations of the leucine incorporation rate have
also been observed in a cell-free system made from rat
liver (Fig. 4). The first point in Fig. 4 is the background
level (marked as zero), which was subtracted from each
of the other values. The radioactivity background was
stable as shown by incorporation kinetics after the
addition of 50 /ig/ml cycloheximide to the cell-free
system (Fig. 4A, lower curve).
Under the conditions of continuous labeling at 30cC,
two or three plateaux were found in the enhanced curve
for cumulative labeling. Some changes in the labeling
40
80
120
Time (min)
Fig. 2. Changes in leucine incorporation into hepatocytes
in organ culture. The samples were taken at 5 min
intervals (<3>
O) a n d 10 min transformations of this
curve. In the latter case, the second time point of the 5
min curve was treated as the starting point, and then every
second point was taken.
120
1100
ao
i
700
300
40
80
120
Time (min)
Fig. 3. Correction (/„,„-) of incorporation level (/) for pool
size (P). Left ordinate (O): I\T\/TlU where T is the total
radioactivity (I+P) at the time points (1) and (i); right
ordinate (A): I/P (%).
rate exceeded the maximum error by a factor of 2-4.
The accuracy of the sampling and the determination of
the protein synthesis rate (maximal error) was determined after changing the temperature of the medium
from 30°C to 4°C or after the addition of 50 jig/ml
cycloheximide to the cell-free system.
366
V. Y. Brodsky and others
0
40
80
120
Time (min)
Fig. 4. Leucine incorporation into proteins of the cell-free
system. Ordinate: radioactivity per sample (in this
experiment, 50 yH). The zero point is the background level,
which was subtracted from each value of protein
radioactivity. (A) Incorporation after continuous labelling
(•); 30 min after transfer of the cell-free system from 30°
to 4°C (*); 10 min after the addition of 50 fjg/ml
cycloheximide (•). (B) Differential curve calculated from
the differences in incorporation between two adjacent time
points (/, and /;.]), thus showing the incorporation rate.
The differential curve demonstrates circahoralian
periodicity. The kinetics of label incorporation shows
rate differences equal to 1500-2000 counts/min (after
background subtraction) and this shows the significance
of maxima and minima even without statistical analysis.
In this experiment, only one sample was obtained at a
certain point. In other experiments, two replicas were
studied for each time-point (Fig. 5). There was good
reproducibility and the plateau values were observed in
the probes containing 2.5 or 4 mg/ml protein per
sample.
Similar patterns were obtained after pulse labeling of
the cell-free system (Fig. 6).
The aa-tRNA fraction of the monolayer also showed
circahoralian periodicity (Fig. 7). The maxima of aatRNA labeling and protein radioactivity coincided in a
given monolayer for 11 periods, whereas in the
remaining four cases these values were in the opposite
phase.
Circahoralian rhythm of the ATP level was also found
in hepatocyte monolayers (Fig. 8). The values obtained
were normalized on the basis of DNA content. Since
adult rat hepatocytes virtually do not divide, DNA
content reflects the number of interphase cells. The
computer program that we used did not allow us to
20
40
Time (min)
Fig. 5. Leucine incorporation into proteins of the cell-free
system. A 2.5 mg sample of protein was used in A while in
B 4 mg was used from the same cell-free system. Duplicate
measurements were made for each point in some series.
40
Time (min)
80
Fig. 6. Pulse labeling (every 10 min) of the cell-free
system. Two sets of samples (O>, A) from the same
system.
show errors in the incorporation curve. The significance
of differences in leucine incorporation rate between
minima (10, 50, 90 min) and maxima (40, 70, 120 min)
was 0.99.
The kinetics of leucine incorporation had a similar
periodicity and was often in the opposite phase to
oscillations of the ATP level. A similar corelation was
found in 14 cases, and in three further cases oscillations
were close to opposite phases. However, in four cases
ATP and protein synthesis rhythms
367
60
40
eo
ISO
Time (min)
Fig. 7. Radioactivity kinetics of the aminoacyl-tRNA
fraction in the hepatocyte monolayer.
40
Fig. 9. Changes in the ATP level in a hepatocyte
monolayer under normal conditions (A) and 1.5 h after
the addition of 1 mM ADP «>)•
2400
1900
1400
40
80
Time (min)
70
SO
120
Fig. 8. Kinetics of the ATP level (nmol ATP/fig DNA) in a
monolayer of hepatocytes (A
A; left ordinate) and the
concomitant incorporation of leucine into proteins of the
same monolayer (^> <^>; right ordinate).
the maxima of ATP concentration and the protein
synthesis rate actually coincided.
The addition of 1 mM ADP to the growth medium
1.5 h before the experiment resulted in a significant
increase in the ATP level in hepatocytes (Fig. 9).
Changes in the ATP level in different specimens of a
given culture were synchronous (see also Fig. 12; and
for the kinetics of leucine incorporation, see Figs 1 and
6). The similarity of oscillations in different samples of a
given culture supports the argument for the biological
nature of such oscillations. Despite the short duration
of the experiment, oscillations of the leucine incorporation rate were observed. The mean incorporation rate,
as well as its rhythm, remained unchanged after an
increase in ATP level (Fig. 10). Similar results were also
observed after 2 mM ADP was added to the medium.
We determined various ATP levels in different
cultures (for comparison see Figs 8 and 12), which could
result from different numbers of cells and different
activities of the cultures. Control and experimental
values may be compared only for the same culture.
The oscillations of the relative incorporation rate of
leucine into proteins were maintained even after the
30
40
Time (min)
80
Fig. 10. Kinetics of the relative leucine incorporation into
cell proteins of the monolayer under normal conditions
(D); and after the addition to samples of the same
monolayer of 1 mM ADP, resulting in a drastic increase in
the intracellular ATP concentration (see Fig. 9). Two
samples were taken at each time point.
overall incorporation rate into the monolayer was
drastically reduced (Fig. 11). The effect was achieved
by adding 0.25 mM ATP to the medium. The
incorporation was equal on average to 770 counts/min,
one hour after such addition, whereas in the control
(the same culture without excess ATP) the incorporation was equal to 1900 counts/min on average for 12
samples. The total monolayer radioactivity (protein
radioactivity plus the pool value) also decreased. This
parameter measures cell permeability to labeled leucine. Interestingly, after the addition of ATP to the
medium the mean concentration of ATP in the cells
showed minimum changes (Fig. 12). The incorporation
of leucine into cell proteins in monolayers was also
decreased even more significantly after the addition of
0.5 mM ATP to the medium.
368
V. Y. Brodsky and others
leo
120
40
0
20
40
Time (mm)
Fig. 11. The relative leucine incorporation into hepatocyte
proteins under normal culture conditions (A) and after the
addition of 0.25 mM ATP «>)•
140
0
20
40
Time (min)
Fig. 12. The ATP level in hepatocytes under normal
culture conditions (A) and after the addition of 0.25 mM
ATP (O).
Discussion
One of the main findings in this study is the periodicity
of the rate of leucine incorporation into hepatocyte
proteins in vitro in monolayer and organ culture. The
periodicity remains after making a correction for the
size of the free amino acid pool in the tissue. Exogenous
leucine added to isolated hepatocytes is known to be
used immediately for protein synthesis and does not
accumulate in pools other than the synthetic one
(Wettenhall and London, 1975). The relative values do
not allow us to measure changes in the actual
incorporation and pool size. In studies of the retina and
salivary gland in vivo and in organ culture (when
specimens could be weighed) it has been demonstrated
that the incorporation rate and pool size oscillate but
not in parallel (Brodsky, 1975).
We do not know the kinetics of changes in the size of
the unlabeled pool in hepatocytes in vitro. When similar
circahoralian oscillations of amino acid incorporation
were studied in sea urchin blastomeres, the pool of
endogenous amino acids was constant (Fry and Gross,
1970). In the cell-free system of hepatocytes that we
have studied the oscillations were similar to those found
in hepatocytes in culture. It should be remembered that
the amino acid concentration is constant in the cell-free
system.
All these data led to the hypothesis that there is a
rhythm of protein synthesis rate in hepatocytes. This
suggestion was made before, on the basis of similar
observations, for certain other cell types (Brodsky,
1975). Observations with a cell-free system provided
further evidence of the periodicity of the protein
synthesis rate. In the cell-free system used by us (see
Eisenstein and Harper, 1984) half the activity is due to
reinitiation of protein synthesis on endogenous mRNA,
and up to 70% of the synthesized proteins are released
from the ribosomes into the medium. Provided the
amino acid pool is constant, periodicity in the incorporation rate into proteins can only be due to the rhythmic
performance of the protein synthesizing machinery.
Oscillations of protein synthesis rate and ATP level in
hepatocytes (occurring often in opposite phases) could
suggest a key role for ATP in the initiation of the
protein synthesis rhythm. This could also follow
rhythmical changes in protein content and respiration
rate in Acanthamoeba. These changes occur in opposite
phases (Lloyd et al., 1982). In gigantic crayfish neurons
as well as the circahoralian rhythm of perikaryon
protein accumulation, there is a similar rhythm of
ATPase activity (Zaguskin, 1986). The hypothesis
suggesting the causal relationship between periodic
oscillations of the ATP level and a similar periodicity of
the protein synthesis rate appears reasonable, because
oscillations of aminoacylation, i.e. the ATP-dependent
process, have a similar periodicity. Oscillations of the
aa-tRNA level were also found in cleaving sea urchin
embryos, in which a circahoralian rhythm of protein
synthesis rate has been detected (Mano, 1975).
However, the relationship between the oscillations of
the ATP level and the protein synthesis rate in
hepatocytes is not stringent. In some cases the high rate
of protein synthesis was not preceded by a high ATP
level. The maximum rate of protein synthesis did not
always correspond with a high level of aa-tRNA. The
aminoacylation of tRNA is not only controlled by ATP.
This process also depends on the activity of pyrophosphatase, pyrophosphorylase and a specific protease
(Tamulevicius et al., 1985; Jacobo-Molina et al., 1988).
Therefore, it is probable that there is aflexiblecoupling
between aminoacylation (including ATP periodic oscillations) and the assembly of the polypeptide.
A non-rigid association between changes in ATP
level on the one hand, and protein synthesis rate on the
other, follows also from the experiments in which
hepatocytes were saturated with ATP. If indeed
oscillations in the ATP level could set the periodicity for
the protein synthesis rate, creation of excess ATP levels
in the cell should lead to the stabilization of protein
synthesis. In our experiments, after the addition of
ADP to the medium, the level of ATP in hepatocytes
increased more than twofold. However, the periodicity
of the protein synthesis rate remained.
ATP and protein synthesis rhythms
The conclusion that ATP pool size is not a pacemaker
for protein synthesis rate follows from the results
obtained with the cell-free system. In such a system an
excess, and a stable level, of ATP (2 mM) are
prerequisites for protein synthesis. Protein synthesis is
also supplied with energy in the form of GTP. In the
cell-free system the level of GTP (1 mM) is also rather
high. This clearly demonstrates the lack of any direct
association between the rhythm or protein synthesis
and the ATP and GTP levels.
Oscillation of protein synthesis was not observed in
some experiments on cell-free systems. It may be
assumed that the incorporation kinetics, oscillating or
linear, depends on the degree of synchronization of
ribosomes isolated from different cells. Oscillations of
protein content have been described in a single cell (for
data and references see Brodsky and Nechaeva, 1988).
The addition of ATP to the culture medium in which
the monolayer was grown markedly decreased leucine
incorporation into proteins without changing the ATP
level in the cells. Far greater changes in the rate of
protein synthesis in the presence of circahoralian
oscillations have been observed in sea urchin blastomeres at low temperature (Mano, 1970, 1975).
Changes in the incorporation as well as in the total
radioactivity present in monolayers appears to be due
primarily to the known effect of exogenous ATP on cell
membranes (Gordon, 1986). It is interesting that
oscillations of the protein synthesis rate are maintained
even after substantial reductions in the level of leucine
incorporation. Moreover, such a decrease in incorporation takes place while the ATP level in the cells
remains stable.
Circahoralian periodicity, just like other biological
rhythms, could be a mechanism for homeostasis, i.e.
the maintenance of a particular level of a substance by
feedback control. Concerning a significant irregularity
in the protein synthesis rhythm and other circahoralian
oscillations, this may result from superimposition of
several regular rhythms (characteristic of diferent cell
types or different proteins present in the same sample).
However, irregular patterns may be due to a certain
inherent lack of organization of the cell patterns,
similar to the fractals that are well known in physics
(Mandelbrot, 1983) and have been recently analysed in
biological systems (Goldberger and West, 1987). Wellknown examples of fractal geometry in nature are
clouds, mountains, contours of the coastline and tree
crowns. In physiology an example of fractals has been
described in the case of the frequency of the heart rate
in man. It should be pointed out that among several
known components of cardiac rhythm the circahoralian
component is as irregular as the rhythm of protein
synthesis and other circahoralian oscillations.
In the case of fractals, oscillations are viewed simply
as a form of existence of a process. Such oscillations will
not be stable in time, and they do not need to have a
pacemaker. Attempts to find a pacemaker for intracellular circahoralian rhythms have not met with
success. One of most remarkable features of circahoralian rhythms is their extreme irregularity: in one
369
experiment, periods with durations varying from 30 to
100 min can be observed and their amplitudes can vary
considerably. The possible use of such kinetics not only
for the maintenance of the average level, but also for
the search for optimum values and the adaptation of
cells to the varying environment is an intriguing
possibility. Under these conditions the rhythm of
energy-yielding processes would affect the rhythm of
the protein synthesis rate - its phase, periods and
amplitude - and even its ability to accomplish protein
synthesis (at very low levels of ATP and GTP);
however, the oscillatory kinetics would remain unaffected.
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(Received 17 December 1991 - Accepted, in revised form,
15 June 1992)