|
Physiol. Chem. Phys. & Med. NMR (2000) 32:167-178 V.
V. Matveev Institute of Cytology, Russian Academy
of Sciences, Abstract: Actin filaments are certainly believed to function as an intracellular signalling system; however, this is not confirmed by direct evidence. We used a two-layer actomyosin gel with a concen-tration gradient of the troponin-tropomyosin complex (TT-complex, Ca2+-sensitive system) between the two layers. To prepare one layer of the system, natural actomyosin (nαν) rich in TT-complex was used. To prepare the second layer, we used desensitized actomyosin (dAM) without the complex. All experimental studies were made in medium with a low ionic strength. Two phenomena were ob-served: (1) dAM blocks Ca2+-sensitivity of nAM when the dAM weight portion in the system (as well as in mixed nAM+dAM suspension) reaches 40% and more; further increase of the dAM portion does not affect the Ca2+-sensitivity; (2) it was electrophoretically shown that a rapid diffusion of the TT-complex from nAM gel into the dAM gel took place. The apparent diffusion coefficient for the TT-complex in dAM gel is about (1-4)10-4 cm2/sec, i.e. three orders higher than the same values for pro-tein diffusion in water.
THE CYTOSKELETON and, particularly, actin filaments are
believed to function as an intracellular
signalling system (by signal we mean any cellular
event which has a regulatory significance). Edelman (1) was the first to
suggest participation of actin filaments in the transfer of signals from the
plasma membrane to the cytoplasm and nucleus.
To elucidate the mechanism
of transfer of a signal related to elements of the cytoskeleton, experiments
with protein model systems reproducing some important features of the cytoplasm can be useful. Such a convenient
model system can be actomyosin. Natural acto-myosin (nAM) contains a
regulatory troponin-tropomyosin complex (TT-complex), whose content may be
easily changed by washing out actomyosin with a solution of a low ionic strength
and alkaline pH. After
removal of the TT-complex, the actomyosin superprecipitation by addition of MgATP
becomes Ca2+-independent, i.e., actomyosin becomes desensi-tized (dAM).
Addition of the TT-complex to dAM results in recovery of Ca2+-sensitivity
(2). Therefore, after removal of the TT-complex from actomyosin, its binding
sites still are able to bind again the regulatory complex. Thus, the use of
actomyosin preparations with different contents of TT-complex can produce gels
with a concentration gradient of the actin-binding proteins.
It should be emphasized
that the TT-complex is strongly bound to actin filaments at physiological ionic strength and pH and serves as a
Ca2+ trigger of muscle contraction (3,4). Hence, the function and
distribution of this regulatory complex can be evaluated not only by analysis
of protein content in various areas of the gel, but also by measurement of its
func-tional parameter, Ca2+-sensitivity of the reaction of
actomyosin superprecipitation. The goal of the present work was to study the interaction of actomyosin gels with differ-ent contents of TT-complex to detect possible redistribution of the TT-complex as a result of its diffusion, translocation, transport, etc.
Materials and
Methods
Isolation of natural
actomyosin (nAM) from rabbit skeletal
muscles was performed at 4-6C,
as
described by Haga et al. (5), with minor modifications. The time of the extraction was 15-20 h. The actomyosin gel (about 30 mg/ml of protein)
with 50 mM KC1 was diluted by the same volume of cold glycerol and stored at
-20C (6). Prior to the experiments, the glycerinated actomyosin was washed
free of glycerol with a solution containing 90 mM KCl
and 14 mM Na-K-phosphate buffer, pH
6.7. After two washings by tenfold volumes of this phosphate buffer (PB),
nAM
was
dissolved
in
0.5 M KC1 and centrifuged for 20
min at 10,000 g.
The
supernatant was
diluted with 9 volumes of cold deionized water, and the col-lected precipitate
was washed twice with PB and then resuspended in PB.
Preparation of desensitized
actomyosin (dAM) was done by washing nAM at 4-6C in pure water
containing a minimal amount of Tris-buffer, pH 8.5-9.0 (2). 15-20 ml aliquots
of the
stored
glycerolized actomyosin preparation
were placed into every 100-ml centrifuge test
tube, and water was added up to the volume of 70 ml. After
the second or third washing, with
centrifugation for 20 min at 10,000 g, the
actomyosin gel swelled (as a result of desali-nation)
to
such a degree that it could no longer be precipitated.
After this step, prior to every centrifugation, 1-2 drops of 3 M KC1 were added
to every tube so that the actomyosin gel volume was about 50% of the suspension
volume after centrifugation. After each centrifu-gation, the supernatant was
replaced by fresh pure water. This procedure was repeated 10 times. The above
procedure was confirmed electrophoretically to result in the nearly com-plete
removal of the TT-complex and in the complete elimination of the Ca2+-sensitivity.
The sensitivity
was
effectively restored by addition of the TT-complex
isolated as described by
Spudich
and Watt (7) to such dAM preparations. After washing, dAM
was centrifuged in 0.5 M KC1 and precipitated by dilution with 10 volumes of
water. The precipitate was washed twice with PB and then resuspended in the
third portion of the buffer.
After addition of MgATP to the actomyosin suspension, the
light scattering began to fall, reaching the minimum steady-state level after
1-2 min (for nAM) or after 5-7 min (for dAM). For nAM
preparations, the decreasing value of the light scattering varied depending on the
presence of Ca2+ or EGTA in the solution. This sensitivity to Ca2+
(Sca) was ex-pressed quantitatively
as a light scattering decrease (LSD):
LSD =I0-I, in the presence both of 0.5 mM Ca2+
(LSDca) and of 0.5 mM EGTA (LSDegta), where I0
is the initial and I, the final level of the light
scattering (in arbitrary units). So Sca = LSDca/LSDegta.
As a rule, three measurements of Sca were done for each actomyosin
sample.
The Sca values
for dAM preparations were close to 1, which indicates that the superpre-cipitation of these
preparations did not depend on Ca2+ and, accordingly, that no
significant amount of the regulatory TT-complex remained in dAM.
The Sca values for nAM prepara-tions were more than 1. This
method of measurement of Ca2+-sensitivity is described in de-tail in
our earlier work (6).
Electrophoresis in polyacrylamide gels in
the presence of sodium dodecyi sulphate (disc-SDS-electrophoresis) was
performed by the method of Sheludko (8), with some modifica-tions for
block-electrophoresis. The gel components were from Reanal (Hungary). Their
composition in the separation gel, unless otherwise specified, was the
following: 0.43 M Tris-HCl (tris(oxymethyl)aminomethane), pH 8.9, 12%
acrylamide (AA), and 0.3% N,N'-methylene-bis-acrylamide
(MBA), in the concentration gel:
0.05 M Tris-HCl, pH 6.8, 2.5% AA, 0.6% MBA, 20% sucrose, and 2% sodium dodecyl
sulphate. The electrode solutions contained 0.6 M glycine and 0.033 M
Tris-buffer, pH 8.3; sodium dodecyl sulphate was added to the upper electrode
solution up to a concentration of 0.1%. Actomyosin samples were solubilized for 5 min in a boiling water bath in a
solution containing 50% glycerol, 5% sodium
dodecyl sulphate, 50 mM
dithiothreitol (Serva, Germany), and 25 mM EDTA (ethyienediaminetetraacetic
acid), pH 7.0. The gels were fixed by a mixture of ethanol-σξ3σοοξ-ξ2ο (1:3:6) and then stained
with 0.125% Coomassie brilliant blue G-250 (Serva,
Germany) dissolved in the fixation
mixture. After the dye excess had been removed from
the gels with 7% acetic acid, densities of
protein bands were measured three times, us-ing an Elscript-3 densitometer
(Hirschmann, Germany). The relative contents of TT-complex proteins were
expressed as percentage to actin.
Formation of two-layer gels. To form an actomyosin gel with non-uniform distribution of the TT-complex, a two-layer system was prepared in 10-ml centrifuge tubes (Figure 1), us-ing the following steps. First, 30 mg of the dAM in suspension were placed in each tube and precipitated by centrifugation for 20 min at 10,000 g (with the final protein concentration of 30-40 mg/ml in the precipitate). Then the supernatant was removed; different amounts of the nAM suspension were added very carefully, and the centrifugation was repeated. The second centrifugation produced a two-layer system (Figure 1) with the overlying supernatant that remained in the tubes during the entire experiment. In order to test deformability of the interlayer surface during the second layer formation, the surface of the first layer was marked with the dye neutral red or with colloidal carbon. No deformation was detected after the second centrifugation. So the possibility of a mixture of different actomyosin preparations can be ruled out.
FIGURE 1.
A diagrammatic sketch of the two-layer actomyosin
systems used, each with a differ-ent ratio of natural (nαν) and desensitized (dAM)
actomyosin. The sizes of the layers are overesti-mated to better demonstrate the
arrangement of the experiments.
In separate experiments,
it was found that the nAM layer (in the two-layer system with the nAM/dAM
ratio 60:40, respectively) lost its Ca2+-sensitivity as early as
after 1-3 min. But to insure the steady-state conditions, we used an overnight
incubation both for the two-layer
systems and for
two-component (n- and dAM) suspensions.
However, the
appar-ent diffusion coefficient was determined during much shorter time
periods.
After formation of
the two-layer systems, the
tubes were chilled for 15-20 h at 4-6C be-fore the
electrophoresis and superprecipitation. After incubation, minimal, about 2 mg,
samples of actomyosin were carefully taken exactly from the surface of the
upper layer of the
two-layer
system for
measurements of the Ca2+-sensitivity and electrophoretic analysis.
The stock
nAM
and dAM preparations used to form
the two-layer system served as control.
To determine the apparent
diffusion coefficient (ADC) for the TT-complex (tropomyosin was used as
a
marker of the complex), the
nAM-dAM interaction was performed in
thin polyvinyl chloride tubes that were chosen so that the outside diameter of
one tube corre-sponded to the inside diameter of the other. This made it
possible to form a very tight, mechanically strong connection. Tubes with the
internal diameters of 3 and 6 mm were used; they formed 2-3-cm-long gels; one end of
each tube was sealed.
The thin tubes were filled
with nAM, the thick ones, with dAM. The filling of the
tubes with actomyosin gel was carried out by the same centrifugation procedure,
as in the prepa-ration of the two-layer systems (10,000 g, 20 min). After
formation of the precipitate, the tubes were cut at the level of the
precipitate/supernatant line and connected to each other very carefully. The
protein contamination from the surface of the joined tubes was thor-oughly
removed.
Several joined tubes were
then incubated at 4-6C. These two-tube systems were quickly frozen
on ice at -20C after different incubation time (from 3 min to 1 h). Then the
thin and thick tubes were separated into two sections, left and right,
respectively, 1 mm from
the point of gel contact. The segment
isolated after these first two cuts was discarded.
The separated tubes were cut into 5
mm slices (resulting in 4-6 segments). After each cut, the cutting tool was cleaned of
protein contamination. Protein from each segment was solubilized and subjected
to electrophoresis. This allowed for the detection of the time-dependent tropomyosin decrease in the nAM gel and
the tropomyosin increase in dAM. The TT-complex diffusion was
studied at a nAM/dAM weight
ratio 1:4. The ADC was calculated as
described by Chang (10).
Statistics. Results of the
experiments were expressed as means + SEM. The differences were considered
statistically significant at P < 0.05, using Student's t-test.
Results
Figure 2 (curve A) illustrates typical results
of the experiments with the two-layer systems. The control nAM
preparation definitely demonstrated a high sensitivity to Ca2+
(curve A, the
left
end, 0% of dAM), while the
other control preparation, dAM, was completely desensitized
(curve A, the right end, 100% of dAM). In the two-layer
system, an increase in the proportion
of
the dAM weight within narrow limits
from 20 to 40% (20% range) resulted in the complete loss of Ca2+-sensitivity
in nAM (the top layer).
FIGURE 2. The Ca2+-sensitivity of the natural actomyosin (nAM), A, arranged as the upper part of the two-layer system (See Figure 1), and the Ca2+-sensitivity of the two-component suspension mixtures (1 mg of protein per ml) of nAM with desensitized actomyosin (dAM.), B, following the 15-20 h incubation (at 4-6C) plotted as a function of the weight portion of the dAM. The value "0" on the X-axis means nαν preparation alone. The value "100" on the X-axis means dAM prepa-ration alone. The value "1" on the Y-axis means the absence of the Ca2+-sensitivity. The data points are the means + SEM from triplicate assays.
As seen from the shape of
the curve A, the degree of the TT-complex redistribution depends on the weight ratio of the
layers, therefore, diffusion of the minor components is trig-gered by the
weight ratio, rather than by the existence of the gradient alone. This
two-layer system
as
a whole initiates the
protein diffusion at a definite nAM/dAM threshold (critical)
ratio.
This "threshold"
hypothesis is confirmed by the data shown in Figure 3. As seen from Figure 2,
the curve describing the dependence of Ca2+-sensitivity on the
weight portion of dAM (without the points for 0 and 100% dAM
because these points do not reflect interac-tions in
two-layer systems) looks like
a hyperbola. After transformation of the data of Figure 2 into the coordinates of Figure 3, this
dependence becomes linear. The linear char-acter of the relationships
demonstrated by Figure 3 is statistically significant for the two-layer system
at the 95% confidence level (correlation coefficient = 0.989, P < 0.05) and
for the two-component suspension, at the 99% confidence level (correlation
coefficient = 0.994, P < 0.01). This linearity proves the hyperbolic
character of the relationships seen in Figure 2 for interacting
actomyosin preparations. Therefore, we can regard the X-axis asymptote (see
the legend for Figure 3) for each curve as a corresponding nAM/dAM
threshold ratio. The threshold value for dAM content to initiate
blockage of Ca2+-sensitivity of nAM was calculated to be 18 +
5% for the two-layer system and 9 + 2%, for the two-component suspension.
FIGURE 3. The linear representation of the data from Figure 2 for natural (nαν) + desensitized actomyosin (dAM) preparations in a form of two-layer systems, A, and as two-component suspen-sion, B. The experimental results (except for the data for individual nAM and dAM preparations, in which the studied interaction does not occur) are well fitted by the linear model: W/S = a + bW, where W is the weight portion (in %) of dαν; S is the Ca2+-sensitivity (in arbitrary units) of nAM (as a part of the two-layer system) or of the nαν + dαν suspension mixtures. The parameters of the equation for the suspensions are: W/S = -10.4(+2.7) + 1.18(+0.05) W, correlation coefficient = 0.994 (P-value < 0.01); for the two-layer systems: W/S = -26.4(+6.6) + 1.50(+0.16) W, correlation coefficient = 0.989 (P-value < 0.05). The statistically significant linearity of the relationships indi-cates that the data both for the two-layer systems and for the nαν + dAM suspension mixtures demonstrate a hyperbolic relation between S and W values. If so, it is possible to calculate the X-axis asymptote for each curve in Figure 2 as a value for the threshold nAM/dAM ratio in accor-dance with the relation: Threshold = a/b, where a and b are parameters of the linear model men-tioned above (the absolute value of a is calculated). The following values for the thresholds were obtained: 9+2% for the suspensions, and 18+5% for the two-layer systems (means + SEM). These thresholds are the levels of dAM (in %, w/w) when the redistribution of TT-complex begins in the systems.
As seen from the shape of
the curve A, the degree of the TT-complex redistribution depends on the weight ratio of the
layers, therefore, diffusion of the minor components is trig-gered by the
weight ratio, rather than by the existence of the gradient alone. This
two-layer system
as
a whole initiates the
protein diffusion at a definite nAM/dAM threshold (critical)
ratio.
This "threshold"
hypothesis is confirmed by the data shown in Figure 3. As seen from Figure 2,
the curve describing the dependence of Ca2+-sensitivity on the
weight portion of dAM (without the points for 0 and 100% dAM
because these points do not reflect interac-tions in
two-layer systems) looks like
a hyperbola. After transformation of the data of
Since nαν (the top
layer) lost its Ca2+-sensitivity, the question arises as to whether
the change in the content of the TT-complex actually takes place in this layer
of the two-layer system under the conditions of our experiment. To answer this
question, several two-layer systems with different nAM/dAM ratios
were formed (Figure 1), and the content of the regulatory proteins in
the top layer of the systems was determined (after 10-15 h of incuba-tion) by
the method of electrophoresis. Figure 4 shows that the content of all the
studied components
of
the regulatory
complex fall sharply when the weight proportion of dAM ex-ceeded a
certain threshold.
FIGURE 4.
The tropomyosin, troponin T, and troponin I contents in
the natural actomyosin (nAM) arranged as the top layer of the two-layer
systems (see Figurel), plotted as a function of the weight portion of the
desensitized actomyosin (dAM) in the lower layer after 15-20 h of
incubation (at 4-6C). These data indicate that the TT-complex diffuses from
the nαν to the dAM
layer in the two-layer systems. The value "0" on the X-axis means nAM
preparation alone. The value "100" on the X-axis means dAM
preparation alone. The data points are the means + SEM from triplicate assays.
Figure 2 into the coordinates of Figure 3, this
dependence becomes linear. The linear char-acter of the relationships
demonstrated by Figure 3 is statistically significant for the two-layer system
at the 95% confidence level (correlation coefficient = 0.989, P < 0.05) and
for the two-component suspension, at the 99% confidence level (correlation
coefficient = 0.994, P < 0.01). This linearity proves the hyperbolic
character of the relationships seen in Figure 2 for interacting
actomyosin preparations. Therefore, we can regard the X-axis as-ymptote (see
the legend for Figure 3) for each curve as a corresponding nAM/dAM
thresh-old ratio. The threshold value for dAM content to initiate
blockage of Ca2+-sensitivity of nAM was calculated to be 18 +
5% for the two-layer system and 9 + 2%, for the two-component suspension.
Since nαν (the top
layer) lost its Ca2+-sensitivity, the question arises as to whether
the change in the content of the TT-complex actually takes place in this layer
of the two-layer system under the conditions of our experiment. To answer this
question, several two-layer systems with different nAM/dAM ratios
were formed (Figure 1), and the content of the regulatory proteins in
the top layer of the systems was determined (after 10-15 h of incuba-tion) by
the method of electrophoresis. Figure 4 shows that the content of all the
studied components
of
the regulatory
complex fall sharply when the weight proportion of dAM ex-ceeded a
certain threshold.
With respect to the Ca2+-sensitivity
changes, similar results were also obtained for mixed nAM and dAM
suspensions containing different proportions of dAM (Figure 2, curve B)
(e.g., with
a
final protein concentration of 1 mg/ml, each ml in the
40% dAM mixture would contain
0.4 mg of dAM and 0.6 mg of nAM). Like in the case when the dAM
weight propor-tion in the two-layer systems reached the 40% level, the
two-component mixture com-pletely
lost Ca2+-sensitivity at the same level
of dAM, although it consisted of 60% of nAM containing a significant amount of
the native TT-complex. The cooperative character of the χ curve (Figure
2) might indicate an interaction between the two types of actomyosin
preparations, otherwise the relationship between the Ca2+-sensitivity
and dAM content would have had a linear character.
As
the
TT-complex is the receptor of Ca2+-, the Ca2+-dependence
of the nAM-dAM inter-action was studied. These sets of
experiments were performed as were other experiments with two-component
suspensions but with the following differences: incubation time was 1 h;
incubation medium contained 0.5 mM EGTA or 0.5 mM Ca2+. When Ca2+-sensitivity
of the
preparations with EGTA was measured,
excess Ca2+ was injected about 1-2 sec prior to the MgATP injection. Data plotted in
Figure 5 show that free Ca2+ enhances the inactivation of the Ca2+-sensitive
system (by dAM) but does not affect the regression slope of the
Ca2+-sensitivity versus dAM: the close-to-linear parts
of the curves are almost parallel.
Our measurements showed
that the tropomyosin (in fact, the TT-complex) ADC (appar-ent diffusion
coefficient) varied within the limits of 4.2-4.410-4 cm2/sec
in the presence of Ca2+ (0.5 mM) and within
the limits of 2.7-4.210-4 cm2/sec
in the presence of EDTA (0.5 mM). Thus, it is evident that divalent cations (Ca2+
and Mg2+) do not significantly affect the rate of the tropomyosin/TT-complex diffusion under the conditions of our experiments.
FIGURE 5. The Ca2+-sensitivity of the two-component suspension mixtures (1 mg of protein per ml) of natural actomyosin (nAM) with desensitized actomyosin (dAM) plotted as a function of the weight portion of the dAM in the mixture after one-hour incubation (at 4-6C) in the presence of 0.5 mM EGTA or 0.5 mM Ca2+. When Ca2+-sensitivity of the preparations with EGTA was measured, excess of Ca2+ was injected about 1-2 sec prior to the MgATP injection. The value "0" on the X-axis means 100% nAM content in the suspension. The data points are the means + SEM from triplicate assays.
Discussion
In this connection, of
interest
are
our data using the two-layer gel
(Figure 1). We found a new type of interaction of the contractile proteins both
in two-layer systems and in suspen-sion mixtures. The nAM preparations
studied had a high Ca2+-sensitivity (3.3 arbitrary units) before
interaction with the dAM layer (Figure 2). However, after the nAM
gel con-tacts with the
dAM
gel, the Ca2+-sensitivity
of nAM (the top layer) begins to depend on the dAM/nAM weight ratio in the two-layer
system; this dependence has a threshold and coop-erative pattern:
nAM
completely
loses Ca2+-sensitivity within
the narrow limits of this ratio changes, from 22/78 to 40/60. This suggests
that nAM, the top layer of the two-layer gel, loses the regulatory
TT-complex.
The
amount of
the Ca2+-sensitive complex in the top layer is
actually reduced with an in-crease
in
the amount of
dAM
in
the
system (Figure
4). At
the
dAM/nAM ratio = 4/6,
the tro-pomyosin
amount,
for example, in the top layer decreases more than 3
times. The amount of other regulatory proteins also falls markedly. The
above-mentioned ratio, 4/6, is the limit, after which the actomyosin
preparations lose completely their Ca2+-sensitivity.
The change in the Ca2+-sensitivity
of nAM in the two-layer system has a definite coop-erative character
that indicates that two actomyosin preparations somehow interact with each
other and this interaction spreads far from the line of contact of the two
gels.
It is clear that the nAM-dAM
interaction begins in a zone of contact of the two gels, but then this process
involves the entire system. Thus, the molecular events in the protein sys-tem
studied appear spatially interconnected.
The
mechanism of such changes requires
the ability of the minor components of actomy-osin gel to move inside the gel.
This conclusion is based on the data presented in Figure 4. These data indicate that in
the bulk of the two-layer gel, after the start of contact was made, the spatial
redistribution (diffusion) of the TT-complex molecules begins to be visible as
evidenced by the minor components leaving the top layer of the system (nAM)
to the point where nAM loses its Ca2+-sensitivity. It is
highly probable that the structural basis for the diffusion of the TT-complex is F-actin, because tropomyosin and
the complex strongly bind
The nAM-dAM interaction
also occurs in the two-component suspensions (Figure 2, curve B). It is
seen that with a decrease in the Ca2+-sensitive nAM fraction,
the decrease in the Ca2+-sensitivity of the whole mixture is not
linear; this relationship might indicate a more complex, unexpected
interaction. In fact, as soon as the nAM content falls below 60%, the Ca2+-sensitivity
of the mixture disappears completely.
The interaction of nAM with dAM
in the presence of free Ca2+ causes a greater decrease in the Ca2+-sensitivity
(Figure 5). This is definitely reasonable, considering multiple Ca2+
effects on the properties and structure of contractile and regulatory proteins
and on their complexes (4).
It is well known that Ca2+,
in addition to its effect on the structure and properties of the TT-complex
(14), enhances the cooperativity and changes some other properties of actin
filaments (13,15) as well as reduces by twice or more the actin affinity to the
TT-complex (16).
The
latter effect is of
interest, as the weakening of the F-actin-TT-complex interaction may facilitate
diffusion of the complex. In the presence of EGTA, actin filaments are more
rigid than in the presence of Ca2+ (17); but in the presence of Ca2+,
the regulated actin (F-actin + TT-complex) and F-actin alone become very
flexible (18,19). From our point of view, the flexibility of actin filaments is
one of the factors modulating the rate of the de-crease or loss of the Ca2+-sensitivity
after the beginning of the nAM-dAM interaction: the flexible
filament facilitates the diffusion process and thus accelerates redistribution
of the TT-complex.
It is interesting to compare the
TT-complex apparent diffusion coefficient (ADC) ob-tained in this work for the
condensed actomyosin gel (at 4-6C) with that for diffusion of other substances
in water (at 25C) (10): 1.1010-5, ethanol; 1.1810-5,
urea; 4.610-6, su-crose; 6.910-7, hemoglobin; 13.3l0-7
cm2/sec, myoglobin. It should be noted again that in our
experiments, tropomyosin was used as a protein marker for the TT-complex. It
might well be that the ADC obtained for "tropomyosin" is in fact the
coefficient for TT-complex.
Thus,
the TT-complex ADC measured at
a lower temperature and in much more viscous, almost solid, medium than water
has an ADC value that is one order of magnitude higher than that for small molecules
of ethanol and urea and three orders of magnitude higher than that of hemoglobin and myoglobin.
The diffusion coefficients for other proteins (lysozyme, ovalbumin, bovine serum albumin), and tobacco virus, as well as
for DNA, also appeared to be three orders of magnitude lower (20) than that for
TT-complex. Hence, if the TT-complex diffused in the water inside the
actomyosin gel, its diffusion coefficient would be much lower. In other words,
the mechanism of the fast diffusion does not allow TT-complex to dissociate
from actomyosin. On the other hand, protein diffusion in the cytoplasm (condensed protein system) is much slower than in aqueous solutions. Thus, the ADCs for proteins in cytoplasm are: 1.010-8 cm2/sec, bovine serum albumin (human fibroblasts, at 22C) (21); 5.710-8 cm2/sec, aldolase (Swiss 3T3 cells, at 36C) (22); 2-310-9 cm2/sec, G-actin (embryonic chicken gizzard cells, at 36C) (23); 1.210-7 cm2/sec, myoglobin (muscle fibers, at 22C) (24). As a rule, the protein mobility in the cytoplasm is much slower than in aqueous solu-tions. This indicates that the mobility inside the cell is limited by interaction of diffusible proteins with the cytoplasmic matrix (25).
It
is interesting to discuss which
properties of F-actin can promote the "sliding" diffusion of
regulatory proteins bound to it: (1) tropomyosin bound to actin has an ability
for lateral movement (26-30); (2) F-actin itself can facilitate diffusion of
the TT-complex by sponta-neous
bends
(18,19), a "twist" of monomeric units in
coordination with each other (31), and rotational movements of actin globules
(32).
Our findings seem to
indicate that the actomyosin gel can be a convenient model for studies on
processes of intracellular signalling systems related to actin filaments. This
model (a
protein gel based on F-actin) is most
likely to provide promising opportunities for studies of conditions required
for protein diffusion in condensed protein systems like the cytoplasm. The
phenomenon of the fast diffusion of TT-complex recalls findings of a higher
mobility of cations bound to polyanions in comparison with the their lower
motility in water (for review see 33). These data indicate that the association
does not necessarily lead to a pronounced reduction in mobility.
So the data obtained can
be summarized in the following words. Natural actomyosin en-riched in the troponin-tropomyosin complex interacts
specifically (by a mechanism still un-known)
with desensitized actomyosin free of the
regulatory complex. This results in (i) loss of Ca2+-sensitivity in natural actomyosin
placed both as a layer of the two-layer system and as a component of its
mixture with desensitized actomyosin, and (ii) redistribution of the TT-complex in the
actomyosin gel. The unknown mechanisms of these effects may play an important
role in the living cell.
The author wishes to thank sincerely Dr. A.A. Vereninov for
helpful support of this study. The author is also grateful to Dr. L.Z. Pevzner
for criticism and to Mr. J. Picheny for his assistance.
References 1. Edelman, G.M. (1976) J. Biochem.
(Tokyo) 79:1P. Full text in PDF (140Kb)
Author's remarks made after publication of this article 1. The main phenomenon is illustrated by Fig.2. It is needed to demonstrate a protein-protein interaction by some direct method. If so, it will open a way for further experiments. 2. In our conditions, diffusion of tropomyosin-troponin complex began after 6 hour's period after creation of the two-tube systems and came to an end after 12 hours. So, the diffusion takes up about 6 hours. 3. The value of tropomyosin/actin ratio in dAM has important role for nAM-dAM interaction. 4. Some time (in 20 % of cases) a fresh natural actomyosin has no Ca-sensitivity. All experiments described above were carried out with natural actomyosin Ca-sensitivity of about 3 a.u. The possible explanation of the mechanism |
