©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Roles of Light Chains in the Activity and Conformation of Smooth Muscle Myosin (*)

(Received for publication, October 10, 1995; and in revised form, January 16, 1996 )

Tsuyoshi Katoh (§) Fumi Morita

From the Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The 20-kDa regulatory (LC20) and 17-kDa essential (LC17) light chain subunits could be removed from porcine aorta smooth muscle myosin by the use of trifluoperazine and ammonium chloride. The isolated heavy chain rebound both light chains, resulting in the restoration of native properties. Experiments on reconstitution of the isolated heavy chain with LC17 and/or LC20 showed that both light chains were required for folding into the 10 S conformation and thus for the phosphorylation-dependent filament formation of smooth muscle myosin. However, LC17 was not essential for the phosphorylation-dependent regulation of actin-activated ATPase activity and superprecipitation but was required for full regulation. LC17 and phosphorylated LC20 were found to act as activators, and dephosphorylated LC20 was found to act as a repressor of the motor activities of smooth muscle myosin.


INTRODUCTION

Smooth muscle myosin is composed of two heavy chains, two 20-kDa regulatory light chains (LC20) (^1)and two 17-kDa essential light chains (LC17). LC20 can be phosphorylated at Ser by Ca/calmodulin-activated myosin light chain kinase and the phosphorylation regulates the activity (for review see Sellers(1991)) and conformation (for review see Trybus(1991)) of smooth muscle myosin. Dephosphorylated myosin has a low actin-activated ATPase activity and is unable to support the movement of actin filaments in a motility assay or to generate superprecipitation. The phosphorylation of LC20 activates these motor activities. On the other hand, smooth muscle myosin takes on two different conformations, referred to as the 6 and 10 S forms based on their sedimentation coefficients. The 6 S myosin assembles into filaments under physiological ionic conditions, whereas 10 S myosin does not. Dephosphorylated myosin filaments disassemble into 10 S monomers upon addition of ATP, whereas phosphorylated myosin remains in filament form.

LC20 has been removed from smooth muscle myosin by an antibody affinity column (Trybus and Lowey, 1988) and by gel filtration in the presence of trifluoperazine (TFP) (Trybus et al., 1994). The removal of LC20 markedly increased the ATPase activity in the absence of actin, thus resulting in a decrease in the actin-activated ATPase activity in comparison with that of native phosphorylated myosin (Trybus et al., 1994). The ability to move actin filaments in a motility assay is also decreased by the removal of LC20 (Trybus et al., 1994). In addition, the LC20-deficient myosin is shown to be unable to fold into the 10 S form (Trybus and Lowey, 1988). Furthermore, an expressed heavy meromyosin lacking LC17 is shown to move actin filaments. The movement is significantly slower than that by control heavy meromyosin but is phosphorylation-dependent (Trybus, 1994). However, the effect of the lack of LC17 on the ATPase activity and conformation of myosin has been unknown. For complete understanding of phosphorylation-dependent regulation of smooth muscle myosin, it is also very important to examine whether LC17 is required for the regulation of actin-activated ATPase activity and of conformation.

It has not been possible to remove LC17 completely from smooth muscle myosin (Trybus, 1994). Here we show for the first time that LC17, in addition to LC20, can be completely removed from smooth muscle myosin without denaturation. Reconstitution experiments with the light chains showed that LC17 and phosphorylated LC20 initiated actin-activated ATPase activity and superprecipitation but that dephosphorylated LC20 inhibited them. For the myosin lacking LC17, not only the rate of superprecipitation but also the actin-activated ATPase activity was considerably lower than those for native myosin but were phosphorylation-dependent, indicative of the nonessential nature of LC17 for the phosphorylation-dependent regulation of the motor activities of smooth muscle myosin. However, myosin lacking LC17 could not fold into the 10 S form, indicating the requirement of LC17 for the phosphorylation-dependent conformational transition and filament formation of smooth muscle myosin.


EXPERIMENTAL PROCEDURES

Protein Preparation

Myosin was prepared from porcine aorta smooth muscle as described by Hasegawa et al.(1988, 1990), and the prepared myosin was almost completely dephosphorylated. Phosphorylated myosin was prepared as described previously (Katoh et al., 1995a). LC20 and phosphorylated LC20 were prepared from aorta myosin as described by Katoh and Morita(1993). LC17 was prepared from aorta myosin according to the method of Grand and Perry (1983) with modifications (Yoshida and Yagi, 1988). Two LC17 isoforms of aorta myosin were isolated by DE32 (Whatman) ion exchange chromatography in the presence of 8 M urea (Hasegawa et al., 1988). Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt(1971) and purified by Sephacryl S-300HR (Pharmacia LKB) gel filtration (Katoh and Morita, 1995). The concentrations of myosin, actin, LC20, and LC17 were determined from the absorbance at 280 nm using the absorption coefficients of 0.48, 1.1, 0.20, and 0.31 (mg/ml) cm, respectively. The concentration of isolated heavy chain (LC-deficient myosin) was determined by the method of Bradford (1976) using myosin as a standard. The molecular weights used for myosin, actin, LC20, LC17, and LC-deficient myosin were 500,000, 42,000, 20,000, 17,000, and 426,000, respectively.

Removal and Readdition of Light Chains

Myosin (5 mg/ml) was incubated on ice for 1 h in 0.4 M NaCl, 10 mM EDTA, 2 mM EGTA, 10 mM ATP, 10 mM DTT, 5 mM TFP, 0.05% Triton X-100, and 40 mM imidazole (pH 6.8). Ammonium chloride was added to the solution to 4.5 M, and the mixture was further incubated on ice for 20 min. The incubated mixture (0.5 ml) was loaded onto a Sephacryl S-300HR column (1.25 times 12 cm) equilibrated with 4.6 M ammonium chloride, 5 mM EDTA, 2 mM EGTA, 3 mM ATP, 2 mM DTT, 0.5 mM TFP, 0.05% Triton X-100, and 40 mM imidazole (pH 6.8), and the protein was eluted at 0.9 ml/min at 6-7 °C. The eluted LC-deficient myosin was immediately passed through a Sephacryl S-300HR spun column at 4 °C to exchange its buffer solution for 0.3 or 0.4 M NaCl, 2 mM DTT, and 20 mM imidazole (pH 7.0). The obtained LC-deficient myosin was immediately mixed with LC17 (equimolar mixture of two LC17 isoforms (Katoh et al., 1995b)) and/or LC20 (2 mol each light chain/mol head) in 0.3 or 0.4 M NaCl, 5 mM MgCl(2), 2 mM DTT, and 20 mM imidazole (pH 7.0) on ice. If necessary, unbound light chains were removed with a Sephacryl S-300HR spun column. The structural state (monomeric or oligomeric state) of LC-deficient myosin or the reconstituted myosin was analyzed by gel filtration HPLC in 0.4 M NaCl and 10 mM sodium phosphate (pH 7.0).

Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was carried out using 10% slab gels under the conditions of Porzio and Pearson(1977). The gels were stained with Coomassie Brilliant Blue G-250 or with silver.

Gel Filtration HPLC

Gel filtration HPLC was performed at room temperature using a TSKgel G5000PW column (7.8 times 300 mm) with a TSK guard column PW (6.0 times 40 mm) attached to a Jasco Gulliver series HPLC system. Elution of proteins was carried out at 0.50 ml/min and monitored by absorbance at 225 nm.

Conformation Analysis

Myosin conformation (6 or 10 S form) was verified by gel filtration HPLC as described by Trybus and Lowey (1988). The conformation of dephosphorylated or phosphorylated myosin was examined at various concentrations of NaCl. Myosin (0.15-0.19 mg/ml) in 0.15-0.4 M NaCl, 2.6 mM MgCl(2), 0.1 mM EGTA, 1 mM DTT, and 10 mM imidazole (pH 7.0) was ultracentrifuged for 10 min at 160,000 times g at 4 °C (Hitachi CP-100H ultracentrifuge) in the presence or the absence of 0.1 mM ATP. The supernatant was analyzed by gel filtration HPLC at the same NaCl concentration as above with 1 mM MgCl(2), 0.1 mM EGTA, and 10 mM sodium phosphate (pH 7.2) in the presence or the absence of 20 µM ATP. The retention time of myosin was plotted against the NaCl concentration (Fig. 1). The profile of the 6-10 S conformational transition of dephosphorylated myosin was in agreement with that of gizzard dephosphorylated myosin shown by Cole and Yount(1992). At 0.24 M NaCl in the presence of ATP, dephosphorylated myosin was still in the 10 S conformation, whereas phosphorylated myosin was in the 6 S conformation. On the basis of these results, the conformation of LC-deficient myosin or the reconstituted myosin was analyzed at 0.24 M NaCl in the presence of ATP.


Figure 1: Salt concentration dependence of the 6-10 S conformational transition of myosin as analyzed by gel filtration HPLC. Dephosphorylated (circle,up triangle) or phosphorylated myosin (bullet,) at 0.15 mg/ml in 0.15-0.4 M NaCl, 2.6 mM MgCl(2), 0.1 mM EGTA, 1 mM DTT, and 10 mM imidazole (pH 7.0) in the presence (circle,bullet) or the absence (up triangle,) of 0.1 mM ATP was ultracentrifuged, and the supernatant (50 µl) was analyzed by gel filtration HPLC (TSKgel G5000PW) in the same concentration of NaCl as above, 1 mM MgCl(2), 0.1 mM EGTA, and 10 mM sodium phosphate (pH 7.2) in the presence or the absence of 20 µM ATP. The retention time of myosin was plotted against the NaCl concentration.



Electron Microscopy

Electron microscopy was performed on a Hitachi H-800 electron microscope operated at 75 kV. For observation of myosin molecules, myosin samples were diluted to 10 µg/ml in 70% glycerol and 0.4 M ammonium acetate, sprayed onto freshly cleaved mica, and rotary-shadowed with platinum at an angle of 6 ° as described previously (Tyler and Branton, 1980; Winkelmann et al., 1983). For observation of myosin filaments, 10 µl of each myosin sample (80-90 µg/ml) in 0.12 M NaCl, 5 mM MgCl(2), 1 mM ATP, 0.1 mM EGTA, 1 mM DTT, and 20 mM imidazole (pH 7.0) was placed on a glow-discharged collodion carbon-coated copper grid and was negatively stained with 1% uranyl acetate as described by Trybus and Lowey(1987).

ATPase Assays and Superprecipitation

ATPase reaction was carried out with 0.12-0.18 µM myosin in 0.1 M NaCl, 6 mM MgCl(2), 1 mM ATP, 0.1 mM EGTA, 1 mM DTT, and 20 mM imidazole (pH 7.0) at 25 °C in the presence and the absence of 20 µM actin. Four aliquots were taken at appropriate time intervals for colorimetric determination of inorganic phosphate (Chifflet et al., 1988; Gonzalez-Romo et al., 1992). Superprecipitation was measured as described previously (Katoh and Morita, 1995).


RESULTS

Removal and Reconstitution of LC20 and LC17 of Smooth Muscle Myosin

Porcine aorta myosin was depleted of its LC20 and LC17 by the method of Trybus et al.(1994) combined with the method of Wagner and Giniger(1981). Myosin was incubated with TFP in the presence of EDTA and ATP to dissociate LC20 followed by further incubation at 0 °C upon addition of ammonium chloride to 4.5 M to dissociate LC17. The myosin heavy chain was separated from the dissociated light chains by Sephacryl S-300HR gel filtration in the presence of TFP and ammonium chloride. The isolated heavy chain (LC-deficient myosin) was essentially free from both LC20 and LC17 (Fig. 2, lane 2) and could rebind stoichiometric amounts of LC17 and/or LC20 (Fig. 2, lanes 3-5).


Figure 2: Characterization of LC-deficient and reconstituted myosins by SDS-polyacrylamide gel electrophoresis. The LC-deficient myosin was mixed with LC17 and/or LC20 (2 mol each light chain/mol head) in 0.4 M NaCl, 5 mM MgCl(2), 2 mM DTT, and 20 mM imidazole (pH 7.0). After unbound light chains were removed by a Sephacryl S-300HR spun column, the myosin samples were analyzed by SDS-polyacrylamide gel electrophoresis. Lane 1, Native myosin; lane 2, LC-deficient myosin; lane 3, myosin reconstituted with LC17; lane 4, myosin reconstituted with LC20; lane 5, myosin reconstituted with LC17 and LC20. HC, myosin heavy chain.



Characterization of LC-deficient and Reconstituted Myosins

The structural states of LC-deficient and reconstituted myosins in high salt conditions were examined by electron microscopy. The rotary-shadowed images of the LC-deficient myosin and LC-deficient myosin reconstituted with LC17 alone showed oligomers in addition to monomers (not shown) as reported for LC-deficient skeletal muscle myosin (Lowey et al., 1993). The heads in monomeric LC-deficient myosin appeared to be more rounded and shorter than in native myosin and the two heads in some LC-deficient myosin monomers appeared to cohere. In contrast, the myosins reconstituted with LC20 alone and with both LC20 and LC17 were observed to be predominantly in monomers, and the shapes of their heads appeared to be indistinguishable from that of native myosin (Fig. 3). The observation that the lack of LC20 resulted in the aggregation of myosin molecules was supported by gel filtration HPLC in 0.4 M NaCl (data not shown).


Figure 3: Characterization of reconstituted myosin by electron microscopy. Native myosin (A), LC-deficient myosin reconstituted with LC20 (B), or with LC17 and LC20 (C) was rotary-shadowed with platinum. Scale bar, 0.1 µm.



Conformations of LC-deficient and Reconstituted Myosins

The conformations of LC-deficient and reconstituted myosins were examined by gel filtration HPLC in 0.24 M NaCl in the presence of ATP as described under ``Experimental Procedures.'' Dephosphorylated and phosphorylated myosins remained soluble, existing predominantly as 10 and 6 S monomers, respectively, and could be separated by gel filtration HPLC ( Fig. 1and Fig. 4, A and B). Most of the LC-deficient myosin and myosins reconstituted with LC17 alone and with dephosphorylated or phosphorylated LC20 alone were pelleted by ultracentrifugation prior to the gel filtration HPLC. These pelleted myosins would be filamentous and unable to fold. Electron microscopy of these myosin samples without ultracentrifugation actually showed the filaments and their aggregates (not shown). The monomers of these myosin species remaining in the supernatant were mainly in the 6 S form (Fig. 4, D-F), but the monomer conformation of LC-deficient myosin was uncertain because of the low concentration of monomers (Fig. 4C). A part of the myosin reconstituted with dephosphorylated LC20 alone was eluted at the time for 10 S myosin (Fig. 4E). However, the 10 S peak fraction showed the LC17 and LC20 bands in approximately equimolar ratio on SDS gel stained with silver (data not shown). The 10 S peak might be due to a very small amount of LC17 remained in the LC-deficient myosin preparation. The myosins reconstituted with both LC17 and dephosphorylated or phosphorylated LC20 were mostly soluble and predominantly in the 10 and 6 S forms, respectively (Fig. 4, G and H), as observed for native myosin (Fig. 4, A and B). Therefore, these results suggest that both LC17 and LC20 are required for the formation of the 10 S conformation and thus for the phosphorylation-dependent 6-10 S conformational transition of smooth muscle myosin.


Figure 4: Conformation of reconstituted myosin analyzed by gel filtration HPLC. Native dephosphorylated (A) and phosphorylated myosin (B), LC-deficient myosin (C), LC-deficient myosin reconstituted with LC17 (D), dephosphorylated LC20 (E), phosphorylated LC20 (F), LC17 and dephosphorylated LC20 (G), or LC17 and phosphorylated LC20 (H) was analyzed by gel filtration HPLC in 0.24 M NaCl, 1 mM MgCl(2), 20 µM ATP, 0.1 mM EGTA, and 10 mM sodium phosphate (pH 7.2) as described in the legend to Fig. 1.



Filament Formation of LC-deficient and Reconstituted Myosins

The filament formation of the LC-deficient and reconstituted myosins was examined in 0.12 M NaCl in the presence of ATP. These myosin samples were negatively stained and observed by electron microscopy (data not shown). The LC-deficient myosin and myosin reconstituted with LC17 alone both showed large aggregates of filaments as previously shown for the LC20-deficient gizzard myosin (Trybus and Lowey, 1988). The myosin reconstituted with LC20 alone also showed filaments and their aggregates irrespective of the phosphorylation state of LC20, but the aggregates appeared to be smaller and looser than those of LC-deficient myosin and myosin reconstituted with LC17 alone. The myosin reconstituted with both LC17 and dephosphorylated LC20 showed few, but slightly more, filaments than native dephosphorylated myosin. The myosin reconstituted with both LC17 and phosphorylated LC20 showed many filaments like native phosphorylated myosin. These results suggest that both light chains are required for the phosphorylation-dependent filament formation of smooth muscle myosin.

ATPase Activities of LC-deficient and Reconstituted Myosins

The ATPase activities of the LC-deficient and reconstituted myosins were measured in the absence and the presence of actin (Table 1). The activities of the LC-deficient myosin in the absence and the presence of actin were both as low as those of native dephosphorylated myosin and were increased 4-5-fold upon reconstitution with LC17 alone, indicating that LC17 could activate the ATPase activity. The reconstitution with dephosphorylated LC20 in addition to LC17 decreased the ATPase activity to of that of myosin reconstituted with LC17 alone, irrespective of the presence of actin. When dephosphorylated LC20 was replaced by phosphorylated LC20, the ATPase activity in the absence of actin was still one-half of that of myosin having only LC17 but increased 2-fold in the presence of actin; the levels of activities were comparable with those of native phosphorylated myosin. Therefore, dephosphorylated LC20 strongly inhibited the ATPase activity that had been activated by LC17, irrespective of the presence of actin. Phosphorylated LC20 also inhibited the ATPase activity in the absence of actin but activated it in the presence of actin. Similar effects of LC20 were also observed in the absence of LC17. However, the level of ATPase activity in the presence of actin for myosin having only phosphorylated LC20, as well as that for myosin having only LC17, was considerably lower than those for myosin having both light chains and for native phosphorylated myosin. The activating effects of LC17 and phosphorylated LC20 seemed to be additive for the ATPase activity in the presence of actin, indicating a requirement of both light chains for the generation of full activity. These results suggest that LC17 acts as an activator and dephosphorylated and phosphorylated LC20, a repressor and an activator, respectively, of the actin-activated ATPase of smooth muscle myosin.



The reconstitution with both light chains fully restored the phosphorylation-dependent regulation of the actin-activated ATPase activity (Table 1). When the LC-deficient myosin was reconstituted with LC20 alone, the actin-activated ATPase activity of the reconstituted myosin was phosphorylation-dependent. Therefore, LC17 may not be essential for the phosphorylation-dependent regulation of actin-activated ATPase activity. However, the degree of regulation for myosin lacking LC17 (4.5-fold activation by phosphorylation) was considerably lower than that for myosin having both LC20 and LC17 (20-fold activation by phosphorylation). This was mainly due to the low activity of myosin having only phosphorylated LC20 in the presence of actin, and the activity was complemented by LC17.

Superprecipitations of Acto-LC-deficient and Acto-reconstituted Myosins

Superprecipitation time courses for the LC-deficient and reconstituted myosins were examined. Superprecipitation was not observed up to 60 min for the LC-deficient myosin, myosin reconstituted with dephosphorylated LC20 alone, that with both LC17 and dephosphorylated LC20, or for native dephosphorylated myosin (Fig. 5, a, c, e, and g). When the LC-deficient myosin was reconstituted with both LC17 and phosphorylated LC20, superprecipitaion occurred quickly after the addition of ATP, as did superprecipitation of acto-native phosphorylated myosin, although the maximum level of superprecipitation for the reconstituted myosin was somewhat lower than that for native phosphorylated myosin (Fig. 5, b and h). When the LC-deficient myosin was reconstituted with phosphorylated LC20 alone, superprecipitation occurred but was considerably slow in comparison with that for native phosphorylated myosin, although the maximum level was even higher than that for native phosphorylated myosin (Fig. 5, f). The results indicate that superprecipitation of actomyosin can be regulated by phosphorylation even if the myosin is depleted of LC17. For the myosin reconstituted with LC17 alone, superprecipitation was observed, but the development was very slow (Fig. 5, d), consistent with a low motility for LC20-deficient myosin (Trybus et al., 1994). The results suggest that superprecipitation is activated by LC17 alone and by phosphorylated LC20 alone but that both light chains are required for full activation.


Figure 5: Superprecipitation of acto-reconstituted myosin. Native dephosphorylated (a) or phosphorylated myosin (b), LC-deficient myosin (c), LC-deficient myosin reconstituted with LC17 (d), dephosphorylated LC20 (e), phosphorylated LC20 (f), LC17 and dephosphorylated LC20 (g), or LC17 and phosphorylated LC20 (h) at 0.24 µM with 5.2 µM actin in 0.1 M NaCl, 5 mM MgCl(2), 0.1 mM EGTA, 1 mM DTT, and 20 mM imidazole (pH 7.0) was mixed with 1 mM ATP at 25 °C. The reaction was monitored by absorbance at 660 nm.




DISCUSSION

Removal of LC20 and LC17 from Smooth Muscle Myosin

The removal of LC20 from smooth muscle myosin has been reported (Trybus and Lowey, 1988; Trybus et al., 1994), but the removal of LC17 has not until now been achieved. On the other hand, the removal of both regulatory and essential light chains from skeletal muscle myosin has been done by gel filtration in the presence of 4.7 M ammonium chloride (Lowey et al., 1993), although this method does not dissociate the light chains from smooth muscle myosin (Lowey and Trybus, 1995). The stronger interaction of regulatory light chain with the heavy chain in smooth muscle myosin than in skeletal muscle myosin (Trybus and Chatman, 1993) may have prevented the dissociation of the light chains from smooth muscle myosin. In fact, we could dissociate LC17 from smooth muscle myosin in a high concentration of ammonium chloride after the dissociation of LC20 by TFP. The results are consistent with the observations that the essential light chain exchange in skeletal muscle myosin subfragment 1 lacking regulatory light chain occurs more readily than in myosin (Waller and Lowey, 1985) and that the essential light chain exchange in desensitized scallop myosin is easier than in intact myosin (Ashiba and Szent-Gyorgyi, 1985).

Roles of LC20 and LC17 in Conformational Transition and Filament Formation of Smooth Muscle Myosin

The lack of either LC20 or LC17 made smooth muscle myosin unable to fold into the 10 S form, resulting in the formation of filaments in the presence of ATP (Fig. 4). The impairment of the ability to fold into the 10 S form for myosin lacking LC20 or both light chains might have been due to cohesion of its two heads, as observed for myosin whose two heads were chemically cross-linked (Katoh and Morita, 1995). However, this was not the case for the reconstituted myosin with dephosphorylated LC20 alone because such cohesion of the two heads was not observed for this myosin by electron microscopy. Not only LC20 but also LC17 might be required for the formation of the native structure of the neck region, which is suggested to be involved in the folding into the 10 S form (Katoh et al., 1995a). On the other hand, the 6-10 S conformational transition is modulated by ATP (Fig. 1), suggesting that signals of the binding of ATP to the active site of myosin somehow transmitted to the neck region resulted in the 10 S conformation. Both LC17 and LC20 subunits may mediate such transmission.

Roles of LC20 and LC17 in Motor Activities of Smooth Muscle Myosin

The removal of regulatory and/or essential light chains from skeletal muscle myosin does not significantly decrease the actin-activated ATPase activity (Pastra-Landis and Lowey, 1986; Lowey et al., 1993). On the other hand, the lack of these light chains decreases the motility of skeletal muscle myosin, but the isolated heavy chain still moves actin filaments (Lowey et al., 1993). Skeletal muscle myosin is not regulated and is always in the ``on'' state that corresponds to the phosphorylated state in smooth muscle myosin. In comparison with phosphorylated smooth muscle myosin, the isolated smooth muscle myosin heavy chain showed the actin-activated ATPase activity of less than (Table 1) and no superprecipitation (Fig. 5). Therefore, the skeletal muscle myosin heavy chain may be in the active state, whereas the smooth muscle myosin heavy chain, in contrast, may be in the inactive state. Both LC17 and phosphorylated LC20 are required for smooth muscle myosin to be in the active state ( Table 1and Fig. 5). The role of skeletal myosin light chains is suggested to stabilize the light chain-binding domain as the lever arm for efficient coupling of ATP hydrolysis to motility (Lowey et al., 1993; Waller et al., 1995). In contrast, LC17 and phosphorylated LC20 activate not only superprecipitation but also the actin-activated ATPase activity for smooth muscle myosin. Therefore, the smooth muscle myosin light chains have specific functional roles in the generation of motor activities, in addition to the structural role that is suggested for skeletal muscle myosin.

On the other hand, LC17 may not be essential for the phosphorylation-dependent regulation of motor activities ( Table 1and Fig. 5), in agreement with the phosphorylation-dependent motility of an expressed gizzard heavy meromyosin lacking LC17 (Trybus, 1994). However, the levels of the motor activities of myosin having only phosphorylated LC20 were still considerably lower than those of myosin having LC17 and phosphorylated LC20 ( Table 1and Fig. 5). Therefore, LC17 may be required for full activation and thus for full regulation of the motor activities of smooth muscle myosin.

The three-dimensional structure of the head of skeletal muscle myosin shows that a 10 nm stretch of the alpha-helical heavy chain segment to which light chains are bound extends near the ATPase and actin-binding sites (Rayment et al., 1993). As the head of smooth muscle myosin probably has a homologous structure, the phosphorylation site may be spatially apart from the ATPase and actin-binding sites. The signals of LC20 phosphorylation should be transmitted to the active site through the light chain-binding domain in each head or through interactions between two heads. In either case, our results suggest that the transmission may be essentially mediated by LC20 to generate the phosphorylation-dependent regulation of motor activities in smooth muscle myosin.

In contrast, the regulation of 6-10 S conformational transition through phosphorylation requires both light chains as described above. The phosphorylation-dependent regulation of motor activities is not directly coupled with that of the conformational transition of smooth muscle myosin. Such an uncoupling between regulations in the activity and conformation is also reported by Ikebe et al.(1994).

Interactions among both light chains and the heavy chain may be required for full regulation of the conformation and motor activities of smooth muscle myosin as suggested for Ca-dependent regulation of scallop myosin (Xie et al., 1994). To elucidate the mechanisms of the phosphorylation-dependent regulation and thus of the intramolecular signal transmission in smooth muscle myosin, more detailed studies are undoubtedly required, and the method of removing light chains presented in this paper could be an aid for the purpose.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-11-716-2111 (ext. 3814); Fax: 81-11-706-4924.

(^1)
The abbreviations used are: LC20, 20-kDa regulatory light chain; LC17, 17-kDa essential light chain; DTT, dithiothreitol; HPLC, high performance liquid chromatography; LC-deficient myosin, isolated myosin heavy chain free from both LC17 and LC20; TFP, trifluoperazine.


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