ARCHIVES
OF BIOCHEMISTRY
AND
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BIOPHYSICS
The Contractile Components
Proteins of Smooth Muscle. Properties and of a Ca 2+-Sensitive Actomyosin from Chicken Gizzard
S. DRISKA Departments
(1975)
of Chemistry
AND
D. J. HARTSHORNE
and Biological Sciences, Carnegie-Mellon Pittsburg, Pennsylvania 15231 Received
September
University,
12, 1974
The preparation and characterization of a Ca2+ -sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg2+ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myo,sin ATPase by actin, and the Cal+ sensitivity were confined to the neutral pH region. The subunit composition of the Cal+ -sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of -130,000.
It is now generally accepted that the contraction--relaxation cycle of all muscles is controlled by changes in the intracellular Ca*+ concentration. The molecular mechanism by which the contractile proteins recognize this change has been studied almost exclusively using skeletal muscle, and a considerable amount of data has accumulated (l-5). On the other hand, the mechanism which operates in smooth muscle is not as well documented. When we began our studies with smooth muscle we were unable to confirm earlier reports (6, 7) on the preparation of the regulatory proteins and, therefore, it was necessary to develop alternate methods. We felt that the most logical starting point was a Ca2+ -sensitive actomyosin. Obviously this protein complex would contain the regulatory proteins, and an initial purification would be achieved as many of the soluble protein contaminants would be lost during the preparative procedure. It was discovered, however, that the isolation of Ca2+-sensitive actomyosin was not as simple with smooth muscle (gizzard) as it was with skeletal muscle. A method to prepare
this complex was, therefore, developed which in our hands and using our experimental tissue (chicken gizzard) gave reproducible results. Since the Ca’+-sensitive actomyosin was to serve as the source of the regulatory proteins, our initial objective was to characterize it with respect to its ATPase properties and subunit composition. One earlier observation that we were particularly interested in was the relatively acidic pH optimum for the Mg*+-activated ATPase activity of smooth muscle actomyosin (8). This was puzzling from a physiological point of view and was also of possible practical significance when considering optimal assay conditions. Our results and conclusions are presented below. The temperature dependence of the Mg2+ ATPase activity and the effect of temperature on Ca2+ sensitivity is also presented. The final part of this paper deals with the components of the Ca’+ sensitive actomyosin. In agreement with the recent results of Breme1 et al. (9), we found that no components analogous to skeletal muscle troponin were present. Our initial observations suggest 203
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
204
DRISKA
AND
that a subunit of approximately 130,000 daltons serves a regulatory function in smooth muscle. METHODS
Preparation
of Gizzard Actomyosin
Chicken gizzards, obtained up to 24 h postmortem, were trimmed of the tough inner lining and fascia. The yield of muscle from whole gizzard was about 50%. The muscle was either frozen or was glycerinated as follows: the muscle was minced and 200-g aliquots were suspended in 50% glycerol, 8 mM sodium phosphate (pH 7.0). About 2 ml of this solution were used per g mince. The suspension was left overnight at 4”C, and then drained through cheesecloth. The initial volume of fresh glycerol solution was added, and after mixing the suspension was stored at -20°C. When required for the preparation of actomyosin the glycerinated mince was strained through cheesecloth and washed three times with 2 vol of 10 mM Tris-HCl (pH 7.6). After each wash the mince was strained through cheesecloth. This procedure removed soluble proteins and also some actin and tropomyosin. The mince was blended for 30 s in 10 vol of 0.1 M KCI, 10 mM Tris-HCl (pH 7.6) and centrifuged at 2OOOg for 10 min. The supernatant was discarded, and the floating layer and the residue were subjected to the blending process as above. Usually three cycles of blending were used. When frozen muscle was used this was blended directly with 10 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), and centrifuged at 2000g for 10 min. The floating layer and residue were subjected to two more cycles of blending and centrifugation using the same solvent. The rest of the procedure is the same for both the frozen and glycerinated muscle. The muscle residue was washed with 5 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), 1% Triton X-100 (see Ref. 10). A loose-fitting Teflon-glass homogenizer was used to disperse the muscle evenly. The suspension was centrifuged at 2000g for 10 min and the supernatant discarded. The Triton X-100 washing procedure was repeated. The residue was then washed twice with 5 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6) to remove detergent. Actomyosin was extracted from the muscle residue by stirring with 0.6 M KCl, 1 mM MgCll, 1 mM ATP, 1 mM dithiothreitol, 50 mru NaHCO, (pH 8.5) for 5 min. (It was convenient to add an equal volume of 1.2 M KCl, 100 mM NaHCO,, 2 mM dithiothreitol to the muscle residue, followed by the Mg-ATP and additional 0.6 M KC1 solvent). A final ratio of muscle residue to solvent of about 1:3 (by volume) was used. The suspension was centrifuged at 23,000g for 20 min. The supernatant was left overnight at 4°C and then diluted with 10 vol of distilled water. The precipitate that formed was collected by centrifugation at 2OOOg for 10
HARTSHORNE min. This constituted the actomyosin and was used without further purification. Usually it was suspended and stored in 10 mM Tris-HCl (pH 7.6). The yield of actomyosin was between 10 and 15 mg per g muscle. The point at which the Triton X-100 washing was done was not critical, and sometimes it was done after the actomyosin extraction. Although the final products were similar, the yield of actomyosin was higher when the detergent washing was done before the extraction step.
Extraction
of Actomyosin
at Low Ionic
Strength The method used was essentially that of Huys (11) modified to include the Triton X-106 washing steps. The treatment of the gizzards up to the blending step was as described above. The muscle residue was homogenized for 30 set in a Waring Blendor in 5 vol of 2 mM ATP, 16 mM NaCl, 10 mM Na,HPO,, 2.6 mM NaHIPO, (pH 7.3). After stirring for 2 h at 4’C the suspension was centrifuged at 23,000g for 10 min. The supernatant was clarified by centrifugation at 48,OOOg for 10 minutes. It was then dialyzed overnight versus 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), 0.1 mM dithiothreitol. The precipitate was collected by centrifugation at 15,000g for 10 min and washed with the 1% Triton X-100 solution as above. After washing in 0.1 M KCl, 10 mM Tris-HCl (pH 7.6) the product was stored in 10 mM Tris-HCl (pH 7.6). The low yield (0.5-1.0 mg per g muscle) obtained with this method made it unsuitable for routine use.
Preparation of Thin Filaments Frozen gizzards (Pel-Freez) were treated as in the preparation of actomyosin up to the stage of extraction (i.e., they were blended and washed in the KCl-Tris solvent, and then subjected to the Triton X-100 treatment). The muscle residue, after the last washing step to remove the detergent, was adjusted to 125 mM KCl, 10 mM MgCl,, 2.5 mM ATP, 1 mM dithiothreitol. Solvent of this composition was added to about twice the volume of the original muscle mince. The suspension was mixed well and centrifuged at 23,000g for 10 min. The supernatant was retained and the residue reextracted with the same volume of the ATP solution, followed by centrifugation, 23,000g for 10 min. The two supernatants were combined and centrifuged at 78,OOOg for 20 min. The precipitate was discarded and additional ATP was added to the supernatant to increase the concentration by 1 mM (This step replenished the ATP level reduced by hydrolysis). The solution was centifuged at 180,oOOg for 1 h, and the pellets were homogenized in a glass-Teflon homogenizer with about 7 vol of a solution containing 10 mM MgCl,, 2.5 mM ATP, 1 mM dithiothreitol, 25 mM Tris-HCl (pH 7.6). The homogenate was centrifuged at 78,000g for 20 min and the
Ca’+-SENSITIVE
ACTOMYOSIN
supernatant was taken as the thin filament preparation. Usually it was dialyzed against 0.1 M KCl, 0.5 mM dithiothretiol, 10 mM Tris-HCl (pH 7.6) before use. The yield was about 0.6 mg/g muscle.
Preparation
of Gizzard
Myosin
Preparation
of Actin from Rabbit Muscle
Skeletal
Preparation of Tropomyosin Chicken Gizzard
Dodecyl Sulfate Polyacrylamide Electrophoresis
The method of Fairbanks et al. (18), containing 5.6% acrylamide, was used. the gels was done at 550 nm using a spectrophotometer equipped with a porter. Estimates of protein composition by measuring the area under relevant scan recording.
from
The method used was basically the same as that described earlier (15) with the exception that the tropomyosin was precipitated between the limits of ammonium sulfate saturation of 50 and 60%. An additional purification by chromatography on hydroxylapatite (16) was frequently used.
Preparation of Other Proteins The preparation of natural actomyosin and myosin from skeletal muscle was as described earlier (15). Desensitized actomyosin from gizzards was prepared by the method of Sparrow and van Bockxmeer (17).
Assays of ATPase Activity
Specific ATPase Actiuities
The various ATPase activities are given in Table I. The activities reported in this table are similar to values given earlier by other investigators (19, 20). Clearly the Mg2+ activity was markedly dependent on the presence or absence of Ca2+, i.e., the absence or presence of EGTA. Up to 90% inhibition of the Mgz+ activity was obtained on removing Ca2+. The range of Ca2+ concentration over which activation and inhibition occurred is shown in Fig. 1. Data for skeletal muscle actomyosin are also included in this figure for comparison, and it is apparent that the range of Ca2+ concentration over which the regulatory proteins operate is similar in both the smooth and skeletal muscle systems. The Ca2+ sensitivity of gizzard actomyosin was relatively stable and was only slightly reduced on standing for 8 h at 25°C (c.f. Ref. 21). Normally the actomyosin retained TABLE ATPase ATPase
ACTIVITIES
activity
10 mM MgCl,, 2.5 mM ATP 10 mM MgCl,, 2.5 mMATP, 1 mM EGTA 2.5 mru CaCll, 2.5 mM ATP 2.5 mM CaCIP, 2.5 mM ATP, 0.8 M KC1 1 mM EDTA, 2.5 mM ATP, 0.5 M KC1
’ EGTA, cetic acid)]
0 Other assay conditions mental Procedure.
[ethyliminodi(a-
I
OF GIZZARD
These were clone at 25’C for 30 min. The Mg*+activated ATPase activity was measured in 10 mM MgCl,, 2.5 mM ATP, 25 mM Tris-HCl (pH 7.6). Caz+ sensitivity was measured by assays done in this solution and in a similar solution containing 1 mM EGTA.’ The assays were stopped by the addition of 1 ml of 25% trichloroacetic acid. For the determination of ATPase activities at different pH values the conditions were as given above with the exception that the following buffers were used: 25 mM sodium 2,2’-ethylenedioxybis
giving a gel Scanning of Gilford 2000 linear transwere made peaks of the
RESULTS
Acetone powder was prepared by the method of Barany and B&my (13) with the modification of an additional washing step with 5 vol of 0.4% NaHCO, for 30 min. Actin was extracted from the powder with 20 vol of distilled H,O for 1 h at room temperature and polymerized for 1 h in 0.1 M KCl. The solution was adjusted to 0.6 M KCl, 20 mM Tris-HCl (pH 7.6) (14) and the F-actin sedimented at about 200,OOOg for 1 h. The F-actin pellets were suspended in about 5 vol of 1 mM MgCl, by gentle homogenization in a glass homogenizer.
The
acetate-acetic acid (pH 4-5.8); 25 mM imidazole-HCl (pH 5.9-7.0); 25 mM Tris-HCl (pH 7.4-9.0); 25 mhi glycine-NaOH (pH 8.9-10.3). Other assay solutions were as described previously (15).
Sodium
The method of Bailin and Barany (12) was used with the following slight modifications. Glycerinated muscle was used and the ribonuclease treatment and ion-exchange chromatography steps were omitted. The separation of myosin and actomyosin at an ionic strength of approximately 0.3 was done twice. The yield of myosin was about 6 mg/g muscle mince.
205
OF GIZZARD
ACTOMYOSIN~
Nanomoles P, rng-’ min-’ 18.0 (11.4) 3.8 (+0.4) 10.0 (h5.4) 84.7 (h22.8) 25.1 (h1.9)
were
as given
in Experi-
DRISKA
AND
FIG. 1. The Ca*+ dependence of the Mg2+activated ATPase activity of skeletal and gizzard actomyosin. Skeletal actomyosin, 0.56 mg (8); gizzard actomyosin, 1.75 mg (0). Under these assay conditions (see Experimental Procedure) the apparent binding constant for CaEGTA was taken to be 1.7 x 10’ hK*.
close to its original activity for up to 3 wk at 0°C. If the Triton X-100 washing steps (see Methods) were omitted, the Ca2+ sensitivity was considerably reduced and was frequently not detected. The relatively low level of the K+-EDTA activity was’a reflection of the presence of actin, since with purified myosin a value of about 350’nmoles Pi min-’ mg-’ was obtained. The ATPase activities in the Ca2+ assay media illustrate one of the differences between smooth and skeletal muscle actomyosins, namely the effect of ionic strength on Ca2+ ATPase, activity. In agreement with other workers (22-241, it was found that an increase in the KC1 concentration caused a marked activation of ATPase activity, and at 0.8 M KC1 the activity was an order of magnitude higher than at zero KCl. In contrast, the Ca2+ activity of skeletal muscle actomysin is inhibited at high ionic strengths. When actomysin was made by the low ionic strength extraction procedure (see Methods) a similar pattern of ATPase activities was obtained. pH Dependence
of ATPase Activity
The Mg2+ activity in the presence and absence of Ca2+ at various pH values is shown in Fig. 2. The shape of the curve is similar to that reported earlier (8, 25) for actomyosin from arterial muscle. The pH
HARTSHORNE
dependence is unusual in that two maxima were found; one at pH 5, and the other at approximately pH 10. At both the acid and alkaline maxima it is significant that Ca2+ sensitivity was lost. This is illustrated by the upper part of Fig. 2, where the difference between the Mg2+ and Mg2+ EGTA activities is plotted over the entire pH range. It was only in the pH range of 6-9.5 that appreciable Ca2+ sensitivity was found. A similar curve was obtained when the EGTA concentration in the assay medium was increased to 10 mM. It, therefore, seemed unlikely that the loss of Ca*+ sensitivity, at least at acid pH, was due to a reduction of Ca2+ binding by EGTA. Two obvious explanations for this behavior were suggested; the first was that the regulatory proteins did not function at either extreme pH value, and the second was that the acid and alkaline optima were due to the Mg2+ activity of myosin alone (i.e., not actin moderated), and, therefore, would not be Ca2+ sensitive. The second possibility seemed the most likely and this was confirmed experimentally. In Fig. 3 is shown the pH dependence of the Mg2+ activity for gizzard myosin and striated muscle myosin. Both showed a similar shape, in that acid and alkaline __-~~
‘0 _E ‘E E a z
002
i
004
c
1
I
003
z i 001
FIG. 2. The pH dependence of the Mg*+-activated ATPase activity of gizzard actomyosin in the presence and absence of Ca*+. Mga+ assay solution (0); Mg*+EGTA assay solution (El). Gizzard actomyosin, 1.2 mg. Upper curve represents Cal+ sensitivity over this pH range, and is the difference between the activities in the Mgz+ and Mgz+ EGTA assay media.
CaZ+-SENSITIVE
ACTOMYOSIN
207
OF GIZZARD
Temperature
Dependence Activity
of ATPase
The effect of temperature on the Mg2+ activity of gizzard actomyosin is shown in Fig. 5, in the form of an Arrhenius plot.
001
4
5
6
7
8
9
lo
11
PH
FIG. 3. The pH dependence of the Mg*+-activated ATPase activit.y of skeletal and gizzard myocin. Gizzard myosin, 2.0 mg (m); skeletal myosin, 1.14 mg (0). Basic Mgz+ assay conditions plus 60 mM KCl.
gizzard myosin was higher than that of skeletal myosin, and this situation was reversed in the alkaline region. A consideration of Mg2+ activity usually is confined to actin-moderated systems, e.g., actomyosin. With skeletal muscle actomyosin, the extent of activation by actin is so large that in a plot of Mg2+ activity versus pH the acid and alkaline optima are not noticed, i.e., the curve is dominated by the actin activation. When, however, one considers a s,ystem where the activation of myosin by actin is considerably less, as in the case of gizzard actomyosin, the acid and alkaline optima become more apparent . It might also be suggested from the above results that the pH dependence of Ca2+ sensitivity of the gizzard actomyosin also reflected the extent ot actm activation of myosin. Obviously it is only under conditions where the myosin can be activated by actin that control via the actin filaments can be effective. In Fig. 4, the Mg2+ activity of myosin alone and myosin plus skeletal muscle actin is shown at different pH values. The significant point to make is that actin activation (upper part of Fig. 4) occurs over the same pH range as Ca2+ sensitivity. This confirms that the pH limits of CaZ’- sensitivity are established as a consequence of actin activation of the myosin.
FIG. 4. The pH dependence of the Mg*+ activated ATPase activity of gizzard myosin and gizzard myosin plus actin. Gizzard myosin, 2.0 mg (El); gizzard myosin, 2.0 mg, plus F-actin, 2.5 mg (0). Assay conditions as in Fig. 3. Upper curve represents actin activation over this pH range.
000,
FIG. 5. An Arrhenius plot of the Mg*+ activated ATPase activity of gizzard actomyosin in the presence and absence of Caz+. Mg*+ assay solution (0); Mga+-EGTA assay solution (El). Actomyosin concentrations and assay times were varied to give reliable levels of ATP hydrolysis.
208
DRISKA
AND
The two curves represent assays done in the presence and absence of Ca2+ (i.e., absence and presence of EGTA, respectively). The Mga+ plus trace Ca*+ plot was virtually linear up to about 4O”C, after which a loss of activity was observed. The activation energy for the linear portion of this curve was about 23 kcal mole-‘. The Mg2+ activity in the absence of Ca’+, however, was different. Below 50°C this curve could be approximated by two straight lines with a break occurring about 32°C. The activation energies for these linear portions of the curve were 37 kcal mole-’ and 10 kcal mole-’ for the higher and lower temperature ranges, respectively. At temperatures greater than 50°C the Mg2+ activity was progressively reduced . It is interesting that the Arrhenius plots for gizzard actomyosin are markedly different from equivalent plots done with skeletal muscle actomyosin (26). If one now considers Ca2+ sensitivity over this temperature range, the results are as shown in Fig. 6. This figure was derived from Fig. 5, and represents the ratio of the Mg2+ activities in the absence and presence of Ca2+. (A ratio of 1 indicates that both activities were the same, and, therefore, the Ca2+ sensitivity was zero). The Ca2+ sensitivity was maximum at about 35”C, and was reduced at both lower and higher temperatures. In the lower temperaI
I
FIG. 6. The temperature dependence of the Cap+ sensitivity of gizzard actomyosin. This figure was derived from Fig. 5 and gives the ratio of the Mg’+/ Mg*+-EGTA activities at various temperatures.
HARTSHORNE
ture range (5YJ-35’C) the reduction of Ca*+ sensitivity was not as marked as in the higher temperature range (35”C-56°C) where it decreased rapidly and was lost at about 53°C. Once again this behavior is significantly different from that shown by skeletal muscle actomyosin (26). Components
of the Caz+-Sensitive Actomyosin
The subunit composition of gizzard actomyosin was examined using SDS polyacrylamide gel electrophoresis. To aid in the identification of the various bands, gizzard myosin and gizzard tropomyosin were also studied. Representative gels are shown in Fig. 7. The myosin sample shows bands corresponding to the heavy chains at -200,000 daltons and light chains at -17,000 and 20,000 daltons. These values are similar to those reported earlier (27, 28). Minor components, including actin at -42,000 daltons, were also visible. It was estimated that the contamination by actin represented about 2% of the total protein. Purified tropomyosin showed two components of about equal staining intensity of -39,000 and 36,000 daltons. Similar results have recently been reported by other investigators (9, 29). The Ca2+-sensitive gizzard actomyosin showed the tropomyosin doublet, the myosin heavy and light chains, and a considerably higher proportion of actin. In addition, subunits in the range of lOO,OOO-130,000 daltons were also seen. The banding pattern in this region was variable and one or multiple bands were observed in different preparations; compare gels (c) and (d) (see Discussion). It is interesting that there were no obvious subunits which would correspond to skeletal muscle troponin. Before it can be concluded, however, that these components were absent it must be established that the myosin light chains did not mask additional components. This was done by preparing thin filaments from gizzard and examining their subunit compcisition and ATPase properties with gizzard myosin. On SDS polyacrylamide gels (Fig. 7) the following components were observed: the tropomyosin doublet, actin, and the 130,000-dalton band. A slight contamina-
Ca2+-SENSITIVE ACTOMYOSIN OF GIZZARD
209
FIG. 7. SDS polyacrylamide gels of proteins from chicken gizzard. (a) myosin (40 rg); (b) tropomyosin (3 pg); (c) Caz+-sensitive actomyosin from frozen gizzard (40 rg); (d) Cal+-sensitive actomyosin from glycerinated gizzard (40 rg); (e) thin filaments (20 pg); (f) desensitized actomyosin (40 pg). The direction of migration was from top to bottom. The bottom black line indicates the position of the tracking dye.
tion by myosin heavy chains was apparent, No components of a subunit weight smaller than tropom,yosin were detected. The latter observation is significant since the thin filaments caused an activation of the Mg2+ ATPase activity of gizzard myosin which was markedly Ca2+ sensitive (Fig. 8). It is, therefore, unlikely that additional components analogous to the skeletal troponin subunits were obscured by the band pattern of the myosin light chains. The above results are consistent with the regulatory proteins being located, at least in part, on the thin filaments. This, however, could not be concluded as there existed the possibility that the myosin light chains might be the only regulatory proteins that were required. Since it was not possible to determine this from the data already presented, additional experimental evidence was necessary. It was shown previously that gizzard actomyosin can be desensitized to Ca2+ by trypsin treatment (6, 30) and one approach was to localize the trypsin-sensitive site. In Table II, it is shown that the Ca2+ sensitivity of a mixture of gizzard myosin- and trypsin-
c % oozo-
s” T
FIG. 8. The Mg’+-activated gizzard myosin plus gizzard assay solution (0); Mg*+-EGTA Myosin 1.0 mg.
ATPase activity of thin filaments. MgZ+ assay solution (El).
treated thin filaments was. drastically reduced. Alternatively Ca2+ sensitivity was not altered when the myosin component was subjected to proteolysis by trypsin. This evidence suggests that the regulatory mechanism in gizzard muscle (or some critical part of it) is located on the thin filaments, as in vertebrate skeletal muscle. Another line of reasoning was to examine the SDS pattern of desensitized gizzard
210
DRISKA TABLE ASSAYS OF ATPase
AND
II ACTIVITP Mg*+ ATPase activity (nanomoles P, min-’ mg-’ myosin) + Ca’+
Gizzard myosin + control thin filaments Gizzard myosin + trypsintreated thin filaments
- Ca*+
18.4
8.2
14.7
15.1
a Gizzard thin filaments were incubated with trypsin, 1OO:l weight ratio, for 20 min at 25°C in 50 mM KCl, 10 mM Tris-HCl (pH 7.6). A lo-fold excess of soybean trypsin inhibitor was added to stop proteolysis. The thin filaments were combined with gizzard myosin at 1:l weight ratio in 0.6 M KCI, 10 mM Tris-HCl (pH 7.6), and dialyzed vs 10 mM Tris-HCl (pH 7.6) for the ATPase assays.
actomyosin and to eliminate the observed components as being solely responsible for Ca2+ regulation. A method to prepare CaZ+-insensitive actomyosin from arterial muscle was reported by Sparrow and van Bockxmeer (17)) and we applied this procedure to gizzard actomyosin. The Mg2+ ATPase activity of the product did not respond to changes in the Ca2+ level (as reported in Ref. 17). The SDS pattern is shown in Fig. 7. Myosin heavy chains, actin, tropomyosin, and myosin light chains were the major components. There was no evidence to indicate that the loss of Ca2+ sensitivity was associated with a loss of myosin light chains. DISCUSSION
The preparation of Ca2+-sensitive actomyosin which is described above has, in our opinion, several advantages over the earlier methods (11, 19). It is rapid, and can be completed in less than 2 days, the yield is relatively high and the product is reproducible. It remains to be established how successful this method will be when applied to other smooth muscles, as it cannot be assumed that all smooth muscles are alike. For example, it is possible to prepare a Ca*+ -sensitive actomyosin from vascular muscle after a relatively simple
HARTSHORNE
procedure, similar to that employed in skeletal muscle studies (F. Fuchs, personal communication) whereas, in our experience, this is not possible with gizzard or uterus. Thus, with any given smooth muscle, the methodology must first be established. There are many variables to be considered, but one modification that can be easily tested is the effect of the detergent treatment. This is the key to the success of our method, and we believe it will prove useful to investigators studying different tissues. The pH profile of the Mg2+ activity of smooth muscle actomyosin has been known for some time (8, 20, 25), and it was recognized earlier (8) that ATP hydrolysis by myosin alone might contribute significantly to the over-all picture. We have shown, in fact, that the activity by myosin alone dominates the pH profile. The acid and alkaline maxima were found with both skeletal and smooth muscle myosins. The significant difference between the two myosins was that in the case of skeletal myosin the activation by actin was over 40-fold whereas with gizzard myosin the activation was almost an order of magnitude less. The relatively poor activation by actin of smooth muscle myosin has been commented on previously (20). Because of the low level of actin-moderated ATPase activity the actomyosin pH curve reflected to a large degree the activity due to myosin alone. Thus, it is very unlikely that either the acid or alkaline maxima has any physiological significance. Maximum activation by actin was found in the neutral pH range, which one would expect if it is assumed that only the actinmoderated Mg2+ activity is relevant in uiuo. However, this raises an interesting point. The highest extent of activation by actin ever achieved in our hands was about 5-fold, and Yamaguchi et al. (31) reported about a 4-fold activation. Yet the inhibition by EGTA of the Mg2+ activity of actomyosin can be over 90% (Fig. 6), indicating that an activation of over lo-fold was obtained with the actomyosin complex. This problem might be resolved when better preparative techniques for myosin become available, but until then the ina-
Cal+-SENSITIVE
ACTOMYOSIN
bility to duplicate the same extent of actin activation by using separated components remains a point of some concern. The Arrhenius plots for the gizzard actomyosin are markedly different from those reported earlier for skeletal muscle actomyosin (26). A detailed comparison of the two plots, however, is not justified since the underlying molecular basis is not understood and only a few general comments are in order. The level of ATPase activity for the gizzard actomyosin was lower at all temperatures than the skeletal muscle actomyosin. This might be relevant when considering the nonlinearity of the Mg*+ EGTA curve for gizzard actomyosin. Since the activity was so low, the contribution to the total ATPase activity by myosin alone could be significant. This could also explain the reduction of Ca2+ sensitivity which occurred at the lower temperatures. At these tem.peratures a greater proportion of the total ATPase activity may not be actin moderated and thus the degree of Ca*+ sensitivity would be less. The band pattern of Ca*+-sensitive actomyosin was fairly simple and showed only a few components that could not be correlated with known muscle proteins. The major unidentified component had a subunit weight of about 130,000 daltons and it is tempting to speculate that this is a regulatory protein. In some actomyosin preparations a more complicated band pattern was observed in this region and this probably was the result of proteolysis. In general, less proteolysis was evident in actomyosins prepared from frozen muscle. The presence of proteases in extracts of actomyosin is one of the reasons why the isolation of well-defined proteins has proved difficult. In all the vertebrate striated muscles that have been studied to date the regulatory proteins are confined to the thin filaments. It has been shown, however, that in some invertebrates the myosin light chains function to control muscle activity (32). Thus, in any unknown contractile system there is a possibility of control via either the thin filaments or the myosin moiety, or both. In the case of gizzard actomyosin all of the evidence suggests
OF GIZZARD
211
that regulation is mediated by the thin filaments, and that the myosin light chains do not constitute a complete regulatory mechanism. The reasons for this conclusion are: (1) trypsin treatment of the thin filaments destroys Ca*+ sensitivity, and (2) actomyosin which has been desensitized to Ca2+ still has both light chains. It is important in consideration of the second point to emphasize that this actomyosin was not irreversibly altered but could be resensitized to changes in the Ca*+ concentration by the addition of the proteins that were removed during the desensitization process. This was shown by Sparrow and van Bockxmeer (17) and confirmed by us. Due to the presence of proteases in this extract it was not possible to identify, with any certainty, the “active” components, although on SDS electrophoresis no bands corresponding to myosin light chains were observed. So far our discussion has been restricted to the possibilities of the regulatory system being confined to either the thin filaments or the myosin molecule. As stated above, we favor the first alternative for gizzard muscle, although the possibility that the myosin light chains function as part of the control mechanism cannot be eliminated. The above results indicate that gizzard actomyosin is controlled by proteins located on the thin filament. Since subunits similar to those found in skeletal muscle troponin were absent (the troponin components in chicken muscle are 44,000, 23,000, and 19,000 daltons [33]), the regulatory function must be assigned to a unique protein. We propose that the 130,000-dalton component fulfills this role. Further work with isolated proteins is necessary to confirm this view. ACKNOWLEDGMENTS The authors are indebted to Mrs. L. Abrams for expert technical assistance. This work was supported by Grants HL-09544 and GM-46407 from the National Institutes of Health. REFERENCES 1. EBASHI, S., OHTSUKI, I., AND MIHASHI, Cold Spring Harbor Symp. Quant. 215-223.
K. (1973) Biol. 37,
212 2. 3.
4.
5.
6.
7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
DRISKA
AND
HARTSHORNE,D. J., AND DREIZEN, P. (1973) Colcl Spring Harbor Symp. @ant. Biol. 37,225-234. GREASER, M. L., YAMAGUCHI, M., BREKKE, C., POTTER, J., AND GERGELY, J. (1973) Cold Spring Harbor Symp. Quant. Biol. 37, 235-244. DRABIKOWSKI, W., NOWAK, E., BARYLKO, B., AND DAEIROWSKA, R. (1973) Cold Spring Harbor Symp. Quant. Biol. 37, 245-249. PERRY, S. V., COLE, H. A., HEAD, J. F., AND WILSON, F. J. (1973) Cold Spring Harbor Symp. Quant. Biol. 37, 251-265. EBASHI, S., IWAKURA, H., NAKAJIMA, H., NAKAMURA, R., AND 001, Y. (1966) Biochem. 2. 345, 201-211. CARSTEN, M. (1971) Arch. Biochem. Biophys. 147, 353-357. MURPHY, R. A. (1969) Microuasc. Res. 1,344-353. BREMEL, R. D., SOBIESZEK, A., AND REEDY, M. K. (1974) Fed. Proc. 33, 1466 (Abstr.). SOURO, R. J., PANG, D. C., AND BRIGGS, F. N. (1971) Biochim. Biophys. Acta 245, 259-262. HUYS, J. (1963) Bull. Sot. Roy. Beige Gynecol. Obstet. 33,429-442. BAILIN, G., AND BARANY, M. (1971) Biochim. Biophys. Acta 236, 292-302. BARANY, M., AND BARRY, K. (1959) Biochim. Biophys. Acta 35, 293-309. SPUDICH, J. A., AND WAIT, S. (1971) J. Biol. Chem. 246,4866-4871. HARTSHORNE, D. J., AND MUELLER, H. (1969) Biochim. Biophys. Acta 175, 301-319. EISENBERG, E., AND KIELLEY, W. W. (1974) J. Biol. Chem. 249, 4742-4748. SPARROW, M. P., AND VAN BOCKXMEER, F. M. (1972) J. Biochem. (Tokyo) 72, 1075-1080. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F.
HARTSHORNE H. (1971) Biochemistry 10, 2606-2617. 19. SPARROW, M. P., MAXWELL, L. C., RUEGG, J. C., AND BOHR, D. F. (1970) Amer. J. Physiol. 219, 1366-1372. 20. BARANY, M., BARANY, K., GAETJENS, E., AND BAILIN, G. (1966) Arch. Biochem. Biophys. 113, 205-221. 21. RUSSELL, W. E. (1973) Eur. J. Biochem. 33, 459-466. 22. NEEDHAM, D., AND CAWKWELL, J. (1956) Biochem. J. 63, 337-344. 23. NEEDHAM, D., AND WILLIAMS, J. (1959) Biochem. J. 73, 171-m. 24. WACHSBERGER, P., AND KALDOR, G. (1971) Arch. Biochem. Biophys. 143, 127-137. 25. MURPHY, R. A. (1971) Amer. J. Physiol. 220, 1494-1500. 26. HARTSHORNE, D. J., BARNS, E. M., PARKER, L., AND FUCHS, F. (1972) Biochim. Biophys. Acta 267, 190-202. 27. KENDRICK-JONES, J. (1973) Phil. Trans. Roy. Sot. London Ser. B. 265, 183-189. 28. LEGER, J., AND FOCANT, B. (1973) Biochim. Biophys. Acta 328, 166-172. 29. CUMMINS, P., AND PERRY, S. V. (1974) Biochem. J. 141, 43-49. 30. DRISKA, S. P., AND HARTSHORNE, D. J. (1974) Fed. Proc. 33, 1581 (Abstr.). 31. YAMAGUCHI, N., MIYAZAWA, Y., AND SEKINE, T. (1970) Biochim. Biophys. Acta 216, 411-421. 32. LEHMAN, W., KENDRICK-JONES, J., AND SZENTGY~RGYI, A. G. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 319-330. 33. HITCHCOCK, S. E., HUXLEY, H. E., AND SZENTGY~RGYI, A. G. (1973) J. Mol. Biol. 80,825-836.
OF BIOCHEMISTRY
AND
167, 203-212
BIOPHYSICS
The Contractile Components
Proteins of Smooth Muscle. Properties and of a Ca 2+-Sensitive Actomyosin from Chicken Gizzard
S. DRISKA Departments
(1975)
of Chemistry
AND
D. J. HARTSHORNE
and Biological Sciences, Carnegie-Mellon Pittsburg, Pennsylvania 15231 Received
September
University,
12, 1974
The preparation and characterization of a Ca2+ -sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg2+ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myo,sin ATPase by actin, and the Cal+ sensitivity were confined to the neutral pH region. The subunit composition of the Cal+ -sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of -130,000.
It is now generally accepted that the contraction--relaxation cycle of all muscles is controlled by changes in the intracellular Ca*+ concentration. The molecular mechanism by which the contractile proteins recognize this change has been studied almost exclusively using skeletal muscle, and a considerable amount of data has accumulated (l-5). On the other hand, the mechanism which operates in smooth muscle is not as well documented. When we began our studies with smooth muscle we were unable to confirm earlier reports (6, 7) on the preparation of the regulatory proteins and, therefore, it was necessary to develop alternate methods. We felt that the most logical starting point was a Ca2+ -sensitive actomyosin. Obviously this protein complex would contain the regulatory proteins, and an initial purification would be achieved as many of the soluble protein contaminants would be lost during the preparative procedure. It was discovered, however, that the isolation of Ca2+-sensitive actomyosin was not as simple with smooth muscle (gizzard) as it was with skeletal muscle. A method to prepare
this complex was, therefore, developed which in our hands and using our experimental tissue (chicken gizzard) gave reproducible results. Since the Ca’+-sensitive actomyosin was to serve as the source of the regulatory proteins, our initial objective was to characterize it with respect to its ATPase properties and subunit composition. One earlier observation that we were particularly interested in was the relatively acidic pH optimum for the Mg*+-activated ATPase activity of smooth muscle actomyosin (8). This was puzzling from a physiological point of view and was also of possible practical significance when considering optimal assay conditions. Our results and conclusions are presented below. The temperature dependence of the Mg2+ ATPase activity and the effect of temperature on Ca2+ sensitivity is also presented. The final part of this paper deals with the components of the Ca’+ sensitive actomyosin. In agreement with the recent results of Breme1 et al. (9), we found that no components analogous to skeletal muscle troponin were present. Our initial observations suggest 203
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
204
DRISKA
AND
that a subunit of approximately 130,000 daltons serves a regulatory function in smooth muscle. METHODS
Preparation
of Gizzard Actomyosin
Chicken gizzards, obtained up to 24 h postmortem, were trimmed of the tough inner lining and fascia. The yield of muscle from whole gizzard was about 50%. The muscle was either frozen or was glycerinated as follows: the muscle was minced and 200-g aliquots were suspended in 50% glycerol, 8 mM sodium phosphate (pH 7.0). About 2 ml of this solution were used per g mince. The suspension was left overnight at 4”C, and then drained through cheesecloth. The initial volume of fresh glycerol solution was added, and after mixing the suspension was stored at -20°C. When required for the preparation of actomyosin the glycerinated mince was strained through cheesecloth and washed three times with 2 vol of 10 mM Tris-HCl (pH 7.6). After each wash the mince was strained through cheesecloth. This procedure removed soluble proteins and also some actin and tropomyosin. The mince was blended for 30 s in 10 vol of 0.1 M KCI, 10 mM Tris-HCl (pH 7.6) and centrifuged at 2OOOg for 10 min. The supernatant was discarded, and the floating layer and the residue were subjected to the blending process as above. Usually three cycles of blending were used. When frozen muscle was used this was blended directly with 10 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), and centrifuged at 2000g for 10 min. The floating layer and residue were subjected to two more cycles of blending and centrifugation using the same solvent. The rest of the procedure is the same for both the frozen and glycerinated muscle. The muscle residue was washed with 5 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), 1% Triton X-100 (see Ref. 10). A loose-fitting Teflon-glass homogenizer was used to disperse the muscle evenly. The suspension was centrifuged at 2000g for 10 min and the supernatant discarded. The Triton X-100 washing procedure was repeated. The residue was then washed twice with 5 vol of 0.1 M KCl, 10 mM Tris-HCl (pH 7.6) to remove detergent. Actomyosin was extracted from the muscle residue by stirring with 0.6 M KCl, 1 mM MgCll, 1 mM ATP, 1 mM dithiothreitol, 50 mru NaHCO, (pH 8.5) for 5 min. (It was convenient to add an equal volume of 1.2 M KCl, 100 mM NaHCO,, 2 mM dithiothreitol to the muscle residue, followed by the Mg-ATP and additional 0.6 M KC1 solvent). A final ratio of muscle residue to solvent of about 1:3 (by volume) was used. The suspension was centrifuged at 23,000g for 20 min. The supernatant was left overnight at 4°C and then diluted with 10 vol of distilled water. The precipitate that formed was collected by centrifugation at 2OOOg for 10
HARTSHORNE min. This constituted the actomyosin and was used without further purification. Usually it was suspended and stored in 10 mM Tris-HCl (pH 7.6). The yield of actomyosin was between 10 and 15 mg per g muscle. The point at which the Triton X-100 washing was done was not critical, and sometimes it was done after the actomyosin extraction. Although the final products were similar, the yield of actomyosin was higher when the detergent washing was done before the extraction step.
Extraction
of Actomyosin
at Low Ionic
Strength The method used was essentially that of Huys (11) modified to include the Triton X-106 washing steps. The treatment of the gizzards up to the blending step was as described above. The muscle residue was homogenized for 30 set in a Waring Blendor in 5 vol of 2 mM ATP, 16 mM NaCl, 10 mM Na,HPO,, 2.6 mM NaHIPO, (pH 7.3). After stirring for 2 h at 4’C the suspension was centrifuged at 23,000g for 10 min. The supernatant was clarified by centrifugation at 48,OOOg for 10 minutes. It was then dialyzed overnight versus 0.1 M KCl, 10 mM Tris-HCl (pH 7.6), 0.1 mM dithiothreitol. The precipitate was collected by centrifugation at 15,000g for 10 min and washed with the 1% Triton X-100 solution as above. After washing in 0.1 M KCl, 10 mM Tris-HCl (pH 7.6) the product was stored in 10 mM Tris-HCl (pH 7.6). The low yield (0.5-1.0 mg per g muscle) obtained with this method made it unsuitable for routine use.
Preparation of Thin Filaments Frozen gizzards (Pel-Freez) were treated as in the preparation of actomyosin up to the stage of extraction (i.e., they were blended and washed in the KCl-Tris solvent, and then subjected to the Triton X-100 treatment). The muscle residue, after the last washing step to remove the detergent, was adjusted to 125 mM KCl, 10 mM MgCl,, 2.5 mM ATP, 1 mM dithiothreitol. Solvent of this composition was added to about twice the volume of the original muscle mince. The suspension was mixed well and centrifuged at 23,000g for 10 min. The supernatant was retained and the residue reextracted with the same volume of the ATP solution, followed by centrifugation, 23,000g for 10 min. The two supernatants were combined and centrifuged at 78,OOOg for 20 min. The precipitate was discarded and additional ATP was added to the supernatant to increase the concentration by 1 mM (This step replenished the ATP level reduced by hydrolysis). The solution was centifuged at 180,oOOg for 1 h, and the pellets were homogenized in a glass-Teflon homogenizer with about 7 vol of a solution containing 10 mM MgCl,, 2.5 mM ATP, 1 mM dithiothreitol, 25 mM Tris-HCl (pH 7.6). The homogenate was centrifuged at 78,000g for 20 min and the
Ca’+-SENSITIVE
ACTOMYOSIN
supernatant was taken as the thin filament preparation. Usually it was dialyzed against 0.1 M KCl, 0.5 mM dithiothretiol, 10 mM Tris-HCl (pH 7.6) before use. The yield was about 0.6 mg/g muscle.
Preparation
of Gizzard
Myosin
Preparation
of Actin from Rabbit Muscle
Skeletal
Preparation of Tropomyosin Chicken Gizzard
Dodecyl Sulfate Polyacrylamide Electrophoresis
The method of Fairbanks et al. (18), containing 5.6% acrylamide, was used. the gels was done at 550 nm using a spectrophotometer equipped with a porter. Estimates of protein composition by measuring the area under relevant scan recording.
from
The method used was basically the same as that described earlier (15) with the exception that the tropomyosin was precipitated between the limits of ammonium sulfate saturation of 50 and 60%. An additional purification by chromatography on hydroxylapatite (16) was frequently used.
Preparation of Other Proteins The preparation of natural actomyosin and myosin from skeletal muscle was as described earlier (15). Desensitized actomyosin from gizzards was prepared by the method of Sparrow and van Bockxmeer (17).
Assays of ATPase Activity
Specific ATPase Actiuities
The various ATPase activities are given in Table I. The activities reported in this table are similar to values given earlier by other investigators (19, 20). Clearly the Mg2+ activity was markedly dependent on the presence or absence of Ca2+, i.e., the absence or presence of EGTA. Up to 90% inhibition of the Mgz+ activity was obtained on removing Ca2+. The range of Ca2+ concentration over which activation and inhibition occurred is shown in Fig. 1. Data for skeletal muscle actomyosin are also included in this figure for comparison, and it is apparent that the range of Ca2+ concentration over which the regulatory proteins operate is similar in both the smooth and skeletal muscle systems. The Ca2+ sensitivity of gizzard actomyosin was relatively stable and was only slightly reduced on standing for 8 h at 25°C (c.f. Ref. 21). Normally the actomyosin retained TABLE ATPase ATPase
ACTIVITIES
activity
10 mM MgCl,, 2.5 mM ATP 10 mM MgCl,, 2.5 mMATP, 1 mM EGTA 2.5 mru CaCll, 2.5 mM ATP 2.5 mM CaCIP, 2.5 mM ATP, 0.8 M KC1 1 mM EDTA, 2.5 mM ATP, 0.5 M KC1
’ EGTA, cetic acid)]
0 Other assay conditions mental Procedure.
[ethyliminodi(a-
I
OF GIZZARD
These were clone at 25’C for 30 min. The Mg*+activated ATPase activity was measured in 10 mM MgCl,, 2.5 mM ATP, 25 mM Tris-HCl (pH 7.6). Caz+ sensitivity was measured by assays done in this solution and in a similar solution containing 1 mM EGTA.’ The assays were stopped by the addition of 1 ml of 25% trichloroacetic acid. For the determination of ATPase activities at different pH values the conditions were as given above with the exception that the following buffers were used: 25 mM sodium 2,2’-ethylenedioxybis
giving a gel Scanning of Gilford 2000 linear transwere made peaks of the
RESULTS
Acetone powder was prepared by the method of Barany and B&my (13) with the modification of an additional washing step with 5 vol of 0.4% NaHCO, for 30 min. Actin was extracted from the powder with 20 vol of distilled H,O for 1 h at room temperature and polymerized for 1 h in 0.1 M KCl. The solution was adjusted to 0.6 M KCl, 20 mM Tris-HCl (pH 7.6) (14) and the F-actin sedimented at about 200,OOOg for 1 h. The F-actin pellets were suspended in about 5 vol of 1 mM MgCl, by gentle homogenization in a glass homogenizer.
The
acetate-acetic acid (pH 4-5.8); 25 mM imidazole-HCl (pH 5.9-7.0); 25 mM Tris-HCl (pH 7.4-9.0); 25 mhi glycine-NaOH (pH 8.9-10.3). Other assay solutions were as described previously (15).
Sodium
The method of Bailin and Barany (12) was used with the following slight modifications. Glycerinated muscle was used and the ribonuclease treatment and ion-exchange chromatography steps were omitted. The separation of myosin and actomyosin at an ionic strength of approximately 0.3 was done twice. The yield of myosin was about 6 mg/g muscle mince.
205
OF GIZZARD
ACTOMYOSIN~
Nanomoles P, rng-’ min-’ 18.0 (11.4) 3.8 (+0.4) 10.0 (h5.4) 84.7 (h22.8) 25.1 (h1.9)
were
as given
in Experi-
DRISKA
AND
FIG. 1. The Ca*+ dependence of the Mg2+activated ATPase activity of skeletal and gizzard actomyosin. Skeletal actomyosin, 0.56 mg (8); gizzard actomyosin, 1.75 mg (0). Under these assay conditions (see Experimental Procedure) the apparent binding constant for CaEGTA was taken to be 1.7 x 10’ hK*.
close to its original activity for up to 3 wk at 0°C. If the Triton X-100 washing steps (see Methods) were omitted, the Ca2+ sensitivity was considerably reduced and was frequently not detected. The relatively low level of the K+-EDTA activity was’a reflection of the presence of actin, since with purified myosin a value of about 350’nmoles Pi min-’ mg-’ was obtained. The ATPase activities in the Ca2+ assay media illustrate one of the differences between smooth and skeletal muscle actomyosins, namely the effect of ionic strength on Ca2+ ATPase, activity. In agreement with other workers (22-241, it was found that an increase in the KC1 concentration caused a marked activation of ATPase activity, and at 0.8 M KC1 the activity was an order of magnitude higher than at zero KCl. In contrast, the Ca2+ activity of skeletal muscle actomysin is inhibited at high ionic strengths. When actomysin was made by the low ionic strength extraction procedure (see Methods) a similar pattern of ATPase activities was obtained. pH Dependence
of ATPase Activity
The Mg2+ activity in the presence and absence of Ca2+ at various pH values is shown in Fig. 2. The shape of the curve is similar to that reported earlier (8, 25) for actomyosin from arterial muscle. The pH
HARTSHORNE
dependence is unusual in that two maxima were found; one at pH 5, and the other at approximately pH 10. At both the acid and alkaline maxima it is significant that Ca2+ sensitivity was lost. This is illustrated by the upper part of Fig. 2, where the difference between the Mg2+ and Mg2+ EGTA activities is plotted over the entire pH range. It was only in the pH range of 6-9.5 that appreciable Ca2+ sensitivity was found. A similar curve was obtained when the EGTA concentration in the assay medium was increased to 10 mM. It, therefore, seemed unlikely that the loss of Ca*+ sensitivity, at least at acid pH, was due to a reduction of Ca2+ binding by EGTA. Two obvious explanations for this behavior were suggested; the first was that the regulatory proteins did not function at either extreme pH value, and the second was that the acid and alkaline optima were due to the Mg2+ activity of myosin alone (i.e., not actin moderated), and, therefore, would not be Ca2+ sensitive. The second possibility seemed the most likely and this was confirmed experimentally. In Fig. 3 is shown the pH dependence of the Mg2+ activity for gizzard myosin and striated muscle myosin. Both showed a similar shape, in that acid and alkaline __-~~
‘0 _E ‘E E a z
002
i
004
c
1
I
003
z i 001
FIG. 2. The pH dependence of the Mg*+-activated ATPase activity of gizzard actomyosin in the presence and absence of Ca*+. Mga+ assay solution (0); Mg*+EGTA assay solution (El). Gizzard actomyosin, 1.2 mg. Upper curve represents Cal+ sensitivity over this pH range, and is the difference between the activities in the Mgz+ and Mgz+ EGTA assay media.
CaZ+-SENSITIVE
ACTOMYOSIN
207
OF GIZZARD
Temperature
Dependence Activity
of ATPase
The effect of temperature on the Mg2+ activity of gizzard actomyosin is shown in Fig. 5, in the form of an Arrhenius plot.
001
4
5
6
7
8
9
lo
11
PH
FIG. 3. The pH dependence of the Mg*+-activated ATPase activit.y of skeletal and gizzard myocin. Gizzard myosin, 2.0 mg (m); skeletal myosin, 1.14 mg (0). Basic Mgz+ assay conditions plus 60 mM KCl.
gizzard myosin was higher than that of skeletal myosin, and this situation was reversed in the alkaline region. A consideration of Mg2+ activity usually is confined to actin-moderated systems, e.g., actomyosin. With skeletal muscle actomyosin, the extent of activation by actin is so large that in a plot of Mg2+ activity versus pH the acid and alkaline optima are not noticed, i.e., the curve is dominated by the actin activation. When, however, one considers a s,ystem where the activation of myosin by actin is considerably less, as in the case of gizzard actomyosin, the acid and alkaline optima become more apparent . It might also be suggested from the above results that the pH dependence of Ca2+ sensitivity of the gizzard actomyosin also reflected the extent ot actm activation of myosin. Obviously it is only under conditions where the myosin can be activated by actin that control via the actin filaments can be effective. In Fig. 4, the Mg2+ activity of myosin alone and myosin plus skeletal muscle actin is shown at different pH values. The significant point to make is that actin activation (upper part of Fig. 4) occurs over the same pH range as Ca2+ sensitivity. This confirms that the pH limits of CaZ’- sensitivity are established as a consequence of actin activation of the myosin.
FIG. 4. The pH dependence of the Mg*+ activated ATPase activity of gizzard myosin and gizzard myosin plus actin. Gizzard myosin, 2.0 mg (El); gizzard myosin, 2.0 mg, plus F-actin, 2.5 mg (0). Assay conditions as in Fig. 3. Upper curve represents actin activation over this pH range.
000,
FIG. 5. An Arrhenius plot of the Mg*+ activated ATPase activity of gizzard actomyosin in the presence and absence of Caz+. Mg*+ assay solution (0); Mga+-EGTA assay solution (El). Actomyosin concentrations and assay times were varied to give reliable levels of ATP hydrolysis.
208
DRISKA
AND
The two curves represent assays done in the presence and absence of Ca2+ (i.e., absence and presence of EGTA, respectively). The Mga+ plus trace Ca*+ plot was virtually linear up to about 4O”C, after which a loss of activity was observed. The activation energy for the linear portion of this curve was about 23 kcal mole-‘. The Mg2+ activity in the absence of Ca’+, however, was different. Below 50°C this curve could be approximated by two straight lines with a break occurring about 32°C. The activation energies for these linear portions of the curve were 37 kcal mole-’ and 10 kcal mole-’ for the higher and lower temperature ranges, respectively. At temperatures greater than 50°C the Mg2+ activity was progressively reduced . It is interesting that the Arrhenius plots for gizzard actomyosin are markedly different from equivalent plots done with skeletal muscle actomyosin (26). If one now considers Ca2+ sensitivity over this temperature range, the results are as shown in Fig. 6. This figure was derived from Fig. 5, and represents the ratio of the Mg2+ activities in the absence and presence of Ca2+. (A ratio of 1 indicates that both activities were the same, and, therefore, the Ca2+ sensitivity was zero). The Ca2+ sensitivity was maximum at about 35”C, and was reduced at both lower and higher temperatures. In the lower temperaI
I
FIG. 6. The temperature dependence of the Cap+ sensitivity of gizzard actomyosin. This figure was derived from Fig. 5 and gives the ratio of the Mg’+/ Mg*+-EGTA activities at various temperatures.
HARTSHORNE
ture range (5YJ-35’C) the reduction of Ca*+ sensitivity was not as marked as in the higher temperature range (35”C-56°C) where it decreased rapidly and was lost at about 53°C. Once again this behavior is significantly different from that shown by skeletal muscle actomyosin (26). Components
of the Caz+-Sensitive Actomyosin
The subunit composition of gizzard actomyosin was examined using SDS polyacrylamide gel electrophoresis. To aid in the identification of the various bands, gizzard myosin and gizzard tropomyosin were also studied. Representative gels are shown in Fig. 7. The myosin sample shows bands corresponding to the heavy chains at -200,000 daltons and light chains at -17,000 and 20,000 daltons. These values are similar to those reported earlier (27, 28). Minor components, including actin at -42,000 daltons, were also visible. It was estimated that the contamination by actin represented about 2% of the total protein. Purified tropomyosin showed two components of about equal staining intensity of -39,000 and 36,000 daltons. Similar results have recently been reported by other investigators (9, 29). The Ca2+-sensitive gizzard actomyosin showed the tropomyosin doublet, the myosin heavy and light chains, and a considerably higher proportion of actin. In addition, subunits in the range of lOO,OOO-130,000 daltons were also seen. The banding pattern in this region was variable and one or multiple bands were observed in different preparations; compare gels (c) and (d) (see Discussion). It is interesting that there were no obvious subunits which would correspond to skeletal muscle troponin. Before it can be concluded, however, that these components were absent it must be established that the myosin light chains did not mask additional components. This was done by preparing thin filaments from gizzard and examining their subunit compcisition and ATPase properties with gizzard myosin. On SDS polyacrylamide gels (Fig. 7) the following components were observed: the tropomyosin doublet, actin, and the 130,000-dalton band. A slight contamina-
Ca2+-SENSITIVE ACTOMYOSIN OF GIZZARD
209
FIG. 7. SDS polyacrylamide gels of proteins from chicken gizzard. (a) myosin (40 rg); (b) tropomyosin (3 pg); (c) Caz+-sensitive actomyosin from frozen gizzard (40 rg); (d) Cal+-sensitive actomyosin from glycerinated gizzard (40 rg); (e) thin filaments (20 pg); (f) desensitized actomyosin (40 pg). The direction of migration was from top to bottom. The bottom black line indicates the position of the tracking dye.
tion by myosin heavy chains was apparent, No components of a subunit weight smaller than tropom,yosin were detected. The latter observation is significant since the thin filaments caused an activation of the Mg2+ ATPase activity of gizzard myosin which was markedly Ca2+ sensitive (Fig. 8). It is, therefore, unlikely that additional components analogous to the skeletal troponin subunits were obscured by the band pattern of the myosin light chains. The above results are consistent with the regulatory proteins being located, at least in part, on the thin filaments. This, however, could not be concluded as there existed the possibility that the myosin light chains might be the only regulatory proteins that were required. Since it was not possible to determine this from the data already presented, additional experimental evidence was necessary. It was shown previously that gizzard actomyosin can be desensitized to Ca2+ by trypsin treatment (6, 30) and one approach was to localize the trypsin-sensitive site. In Table II, it is shown that the Ca2+ sensitivity of a mixture of gizzard myosin- and trypsin-
c % oozo-
s” T
FIG. 8. The Mg’+-activated gizzard myosin plus gizzard assay solution (0); Mg*+-EGTA Myosin 1.0 mg.
ATPase activity of thin filaments. MgZ+ assay solution (El).
treated thin filaments was. drastically reduced. Alternatively Ca2+ sensitivity was not altered when the myosin component was subjected to proteolysis by trypsin. This evidence suggests that the regulatory mechanism in gizzard muscle (or some critical part of it) is located on the thin filaments, as in vertebrate skeletal muscle. Another line of reasoning was to examine the SDS pattern of desensitized gizzard
210
DRISKA TABLE ASSAYS OF ATPase
AND
II ACTIVITP Mg*+ ATPase activity (nanomoles P, min-’ mg-’ myosin) + Ca’+
Gizzard myosin + control thin filaments Gizzard myosin + trypsintreated thin filaments
- Ca*+
18.4
8.2
14.7
15.1
a Gizzard thin filaments were incubated with trypsin, 1OO:l weight ratio, for 20 min at 25°C in 50 mM KCl, 10 mM Tris-HCl (pH 7.6). A lo-fold excess of soybean trypsin inhibitor was added to stop proteolysis. The thin filaments were combined with gizzard myosin at 1:l weight ratio in 0.6 M KCI, 10 mM Tris-HCl (pH 7.6), and dialyzed vs 10 mM Tris-HCl (pH 7.6) for the ATPase assays.
actomyosin and to eliminate the observed components as being solely responsible for Ca2+ regulation. A method to prepare CaZ+-insensitive actomyosin from arterial muscle was reported by Sparrow and van Bockxmeer (17)) and we applied this procedure to gizzard actomyosin. The Mg2+ ATPase activity of the product did not respond to changes in the Ca2+ level (as reported in Ref. 17). The SDS pattern is shown in Fig. 7. Myosin heavy chains, actin, tropomyosin, and myosin light chains were the major components. There was no evidence to indicate that the loss of Ca2+ sensitivity was associated with a loss of myosin light chains. DISCUSSION
The preparation of Ca2+-sensitive actomyosin which is described above has, in our opinion, several advantages over the earlier methods (11, 19). It is rapid, and can be completed in less than 2 days, the yield is relatively high and the product is reproducible. It remains to be established how successful this method will be when applied to other smooth muscles, as it cannot be assumed that all smooth muscles are alike. For example, it is possible to prepare a Ca*+ -sensitive actomyosin from vascular muscle after a relatively simple
HARTSHORNE
procedure, similar to that employed in skeletal muscle studies (F. Fuchs, personal communication) whereas, in our experience, this is not possible with gizzard or uterus. Thus, with any given smooth muscle, the methodology must first be established. There are many variables to be considered, but one modification that can be easily tested is the effect of the detergent treatment. This is the key to the success of our method, and we believe it will prove useful to investigators studying different tissues. The pH profile of the Mg2+ activity of smooth muscle actomyosin has been known for some time (8, 20, 25), and it was recognized earlier (8) that ATP hydrolysis by myosin alone might contribute significantly to the over-all picture. We have shown, in fact, that the activity by myosin alone dominates the pH profile. The acid and alkaline maxima were found with both skeletal and smooth muscle myosins. The significant difference between the two myosins was that in the case of skeletal myosin the activation by actin was over 40-fold whereas with gizzard myosin the activation was almost an order of magnitude less. The relatively poor activation by actin of smooth muscle myosin has been commented on previously (20). Because of the low level of actin-moderated ATPase activity the actomyosin pH curve reflected to a large degree the activity due to myosin alone. Thus, it is very unlikely that either the acid or alkaline maxima has any physiological significance. Maximum activation by actin was found in the neutral pH range, which one would expect if it is assumed that only the actinmoderated Mg2+ activity is relevant in uiuo. However, this raises an interesting point. The highest extent of activation by actin ever achieved in our hands was about 5-fold, and Yamaguchi et al. (31) reported about a 4-fold activation. Yet the inhibition by EGTA of the Mg2+ activity of actomyosin can be over 90% (Fig. 6), indicating that an activation of over lo-fold was obtained with the actomyosin complex. This problem might be resolved when better preparative techniques for myosin become available, but until then the ina-
Cal+-SENSITIVE
ACTOMYOSIN
bility to duplicate the same extent of actin activation by using separated components remains a point of some concern. The Arrhenius plots for the gizzard actomyosin are markedly different from those reported earlier for skeletal muscle actomyosin (26). A detailed comparison of the two plots, however, is not justified since the underlying molecular basis is not understood and only a few general comments are in order. The level of ATPase activity for the gizzard actomyosin was lower at all temperatures than the skeletal muscle actomyosin. This might be relevant when considering the nonlinearity of the Mg*+ EGTA curve for gizzard actomyosin. Since the activity was so low, the contribution to the total ATPase activity by myosin alone could be significant. This could also explain the reduction of Ca2+ sensitivity which occurred at the lower temperatures. At these tem.peratures a greater proportion of the total ATPase activity may not be actin moderated and thus the degree of Ca*+ sensitivity would be less. The band pattern of Ca*+-sensitive actomyosin was fairly simple and showed only a few components that could not be correlated with known muscle proteins. The major unidentified component had a subunit weight of about 130,000 daltons and it is tempting to speculate that this is a regulatory protein. In some actomyosin preparations a more complicated band pattern was observed in this region and this probably was the result of proteolysis. In general, less proteolysis was evident in actomyosins prepared from frozen muscle. The presence of proteases in extracts of actomyosin is one of the reasons why the isolation of well-defined proteins has proved difficult. In all the vertebrate striated muscles that have been studied to date the regulatory proteins are confined to the thin filaments. It has been shown, however, that in some invertebrates the myosin light chains function to control muscle activity (32). Thus, in any unknown contractile system there is a possibility of control via either the thin filaments or the myosin moiety, or both. In the case of gizzard actomyosin all of the evidence suggests
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211
that regulation is mediated by the thin filaments, and that the myosin light chains do not constitute a complete regulatory mechanism. The reasons for this conclusion are: (1) trypsin treatment of the thin filaments destroys Ca*+ sensitivity, and (2) actomyosin which has been desensitized to Ca2+ still has both light chains. It is important in consideration of the second point to emphasize that this actomyosin was not irreversibly altered but could be resensitized to changes in the Ca*+ concentration by the addition of the proteins that were removed during the desensitization process. This was shown by Sparrow and van Bockxmeer (17) and confirmed by us. Due to the presence of proteases in this extract it was not possible to identify, with any certainty, the “active” components, although on SDS electrophoresis no bands corresponding to myosin light chains were observed. So far our discussion has been restricted to the possibilities of the regulatory system being confined to either the thin filaments or the myosin molecule. As stated above, we favor the first alternative for gizzard muscle, although the possibility that the myosin light chains function as part of the control mechanism cannot be eliminated. The above results indicate that gizzard actomyosin is controlled by proteins located on the thin filament. Since subunits similar to those found in skeletal muscle troponin were absent (the troponin components in chicken muscle are 44,000, 23,000, and 19,000 daltons [33]), the regulatory function must be assigned to a unique protein. We propose that the 130,000-dalton component fulfills this role. Further work with isolated proteins is necessary to confirm this view. ACKNOWLEDGMENTS The authors are indebted to Mrs. L. Abrams for expert technical assistance. This work was supported by Grants HL-09544 and GM-46407 from the National Institutes of Health. REFERENCES 1. EBASHI, S., OHTSUKI, I., AND MIHASHI, Cold Spring Harbor Symp. Quant. 215-223.
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