Smooth muscle contraction is initiated by phosphorylation of myosin's regulatory light chain (RLC)()(1, 2, 3) . In vitro, this myosin and similar myosin IIs (4) must be phosphorylated to hydrolyze ATP rapidly in the presence of actin and to move actin filaments. Phosphorylation appears to be sufficient to activate smooth muscle myosin, independent of changes in filament assembly(5) . Heavy meromyosin, having two heads but lacking the carboxyl-terminal two-thirds of the tail, is regulated by phosphorylation(6, 7) . Subfragment-1, containing a single head and no tail, is always active (7, 8) . These data suggest that the ``off'' state requires head-head or head-tail interactions. To differentiate between these two possibilities, we have investigated a highly purified single-headed proteolytic fragment isolated from a proteolytic digest of smooth muscle myosin.

Single-headed proteolytic fragments of myosins from various muscles that exhibit different types of regulation have been previously studied. Purified single-headed fragments of unregulated skeletal muscle myosin have been shown to assemble into normal filaments(9, 10, 11) and produce normal isometric tension per head(11, 12) . Cooperative interaction between the heads is not essential to develop sliding force (11) .

To our knowledge, there are no reports of regulatory studies with purified single-headed fragments of myosins that exhibit myosin-linked regulation. However, the question of the role of two heads in regulation has been previously addressed. Scallop S1 is similar to smooth S1 in that it is unregulated(13) . In studies with unpurified protease digestion mixtures of scallop-striated muscle myosin, the single-headed myosin fragment appears to retain regulation (13) by Ca binding to the interface between the regulatory and essential light chains(14) . Similar experiments with unpurified smooth muscle myosin digests suggest that the single-headed myosin lacks phosphorylation-dependent regulation(15) . This result contradicted an earlier report of the insoluble fraction of papain digests of smooth muscle myosin, suggesting that the single-headed form required phosphorylation for actin-activated ATPase(16) . Thus, from these initial studies with unpurified single-headed fragments, it is not clear whether the structural interactions responsible for the two myosin-linked regulatory mechanisms, Ca binding and phosphorylation, are fundamentally different or whether they might be similar.

In our studies of proteolytic digestion of smooth muscle myosin, we found that the single-headed form is a transient intermediate that is never present in large amounts relative to the total number of myosin heads in a mixture. For this reason, it is difficult to study the properties of the single-headed form without further purification. Our approach here was to avoid potential problems of proteolytic digestion mixtures and to first highly purify a single-headed fragment of smooth muscle myosin in preparative amounts. In this way, the single-headed myosin was not contaminated with large amounts of double-headed myosin and S1, normally produced in a proteolytic digest. Isolation of a single-headed fragment in a purified form has allowed an investigation of the phosphorylation-dependent regulatory behavior by steady-state ATPase and in vitro motility measurements.

MATERIALS AND METHODS

Myosin was purified from frozen chicken gizzards (17) obtained from Pell-Freeze. The preparative isolation of single-headed myosin combines aspects from previous single-headed myosin preparations(10, 12, 13, 18) . The pH of all solutions was adjusted at 4 °C. All dialyses and centrifugations were at 4 °C. Freshly purified myosin (400 mg) was digested in filamentous form at 4 mg/ml in 0.2 M ammonium acetate, pH 7.8, 2 mM EGTA, 1 mM DTT with 2 µg/ml papain (Sigma, 20 units/mg protein) for 27 min at 25 °C. After quenching the digestion with 5 µg/ml leupeptin, the soluble heads (S1) were removed by twice centrifuging and washing the myosin filaments with digestion buffer including leupeptin. The resulting pellet, containing double-headed and single-headed myosin and rods, was dialyzed overnight into actin binding buffer (0.35 M NaCl, 10 mM sodium phosphate, pH 7.2, 1 mM MgCl, 1 mM EGTA, 5 mM DTT) and centrifuged (350,000 g, 30 min) to remove insoluble material. F-actin from rabbit skeletal muscle (19) was dialyzed extensively versus 50 mM KCl, 1 mM MgCl, 0.2 mM DTT, 10 mM Tris, pH 8.2, to remove ATP. The actin and the digested myosin were then mixed in a ratio of 0.5 mg of actin to 1 mg of digest (estimated using = 5.6 cm). The final concentrations were 4-5 mg/ml myosin digest and 2-2.5 mg/ml actin. After adjusting the final KCl concentration to 0.3 M, the mixture was centrifuged (170,000 g, 50 min), leaving myosin rods in the supernatant. The pellet was homogenized in actin binding buffer and centrifuged twice more to completely remove rods from the pellet. Single-headed myosin was selectively released from the pellet by homogenizing in actin binding buffer containing 3 mM sodium pyrophosphate and centrifuging as before. Double-headed myosin and actin remained in the pellet. The yield of single-headed myosin ( = 4.3 cm(12) ) could be doubled by repeating the last resuspension and centrifugation. The purification procedure was monitored by nondenaturing gel electrophoresis in the presence of sodium pyrophosphate(20) . Single-head myosin was identified as an intermediate band that transiently appeared in papain digestion time courses, as in the analysis of scallop myosin digests(13) . Gels were scanned with a BioImage Visage 60-110 scanner to estimate purity. The clipped RLC of the single-headed myosin was replaced with exogenously added RLC or tpRLC(21) . Pellets were then resuspended in 15 mM Tris, pH 7.5, at 25 °C, 0.3 M KCl, 5 mM MgCl, 1.0 mM EGTA, 0.1 mM DTT, and the solution was centrifuged to remove insoluble material. A single-headed sample was prepared that was treated identically, except that light chains were not added and the sample remained at 4 °C during the exchange step. A parallel set of double-headed samples was also prepared. Complete exchange was verified by analyzing the samples on a 4-15% polyacrylamide SDS gel (see Fig. 1B) or on a polyacrylamide gel in the presence of urea (21) (data not shown). Electron microscopy was performed on a Hitashi 600 electron microscope operated at 75 kV. Specimens were prepared (22) at 22 µg/ml protein in 66% glycerol, 0.5 M KCl, 20 mM Tris, pH 8.2, 10 mM DTT, 1 mM EGTA.

Figure 1: Characterization of single-headed myosin. A, nondenaturing gel electrophoresis. dh, double-headed; sh, single-headed. Lane1, initial double-headed myosin (7 µg); lane2, after papain digestion and S1 removal (5 µg); lane3, single-headed myosin preparation (3 µg); lane4, same as lane3 except 4.2 µg; lane5, rod fraction (5 µg). B, the extent of exchange of exogenously added RLC into single-headed myosin (and double-headed myosin controls) was monitored by SDS-gel electrophoresis. Lane1, initial double-headed myosin; lane2, after papain digestion and S1 removal; lane3, double-headed undigested control prior RLC exchange; lane4, double-headed undigested control after RLC exchange; lane5, double-headed undigested control after tpRLC exchange; lane6, single-headed myosin prior to adding RLC; lane7, single-headed myosin after RLC exchange; lane8, single-headed myosin after tpRLC exchange. C, platinum-shadowed carbon-coated images of double-headed myosin; D, single-headed myosin exchanged with RLC (samples shown in lanes4 and 7 in B, respectively). Bar = 50 nm.

The in vitro motility assays were performed, and the data were analyzed as described (23) except the motility buffer contained 60 mM KCl, 20 mM MOPS (pH 7.2), 5 mM MgCl, 20 mM DTT, 0.1 mM EGTA, 200 nM gizzard tropomyosin, 2.5 mg/ml glucose, 0.1 mg/ml glucose oxidase, 2 µg/ml catalase, 0.7% methylcellulose, and the assay temperature was 30 °C.

RESULTS AND DISCUSSION

Our approach was to isolate in preparative amounts and subsequently characterize a single-headed fragment isolated from a papain digestion of gizzard myosin. Our single-headed myosin preparation contained 95-96% single-headed myosin and 4-5% double-headed myosin as determined by densitometric scanning of a non-denaturing gel (Fig. 1A, lanes3 and 4). SDS-gel electrophoresis of the single-headed preparation (Fig. 1B, lane6) showed that some of the heavy chains (hc, 200 kDa) were clipped by papain at the junction between the 50- and 20-kDa S1 head segments to generate the carboxyl-terminal 140-kDa and amino-terminal 70-kDa fragments (18, 24) . The 120-kDa rod fragment verifies the presence of single-headed myosin. Approximately 80% of the RLC, clipped between Arg-16 and Thr-17, migrated just below the essential light chain (Fig. 1B, lane6) and could not be phosphorylated by myosin light chain kinase(25) . Therefore, the endogenous clipped RLC was exchanged (21) with exogenously added RLC (Fig. 1B, lane7) or tpRLC (Fig. 1B, lane8). Similar RLC exchange procedures have been used by other investigators who have shown that this method does not significantly alter the ATPase activity and the ability of the myosin to undergo the salt-dependent transition between 6 S and 10 S forms(26, 27) . Gel scanning showed that both double-headed (Fig. 1B, lanes4 and 5) and single-headed (lanes7 and 8) exchanged samples contained the same ratio of native RLC to essential light chain as untreated native myosin (lane1). Electron micrographs showed an intact single-headed myosin (Fig. 1D), which clearly lacks a head domain upon comparison with double-headed myosin (Fig. 1C). Several fields of view were analyzed (data not shown), and this analysis showed that only 1-2% of the myosin in our single-headed preparation was double-headed.

The MgATPase activities of both untreated and RLC-exchanged double-headed myosins were activated with actin upon thiophosphorylation by about a factor of 40 (Table 1). In contrast, single-headed myosins, with either clipped or intact RLC, had high MgATPase activity with actin even without thiophosphorylation. Thiophosphorylation increased this activity only by a factor of about 1.6. These data are consistent with a lack of phosphorylation-dependent regulation in the single-headed myosin.

We measured in vitro actin filament movement as another test for phosphorylation-dependent regulation (Fig. 2). The thiophosphorylated double-headed myosin moved actin filaments at about 1.4 µm/s (Fig. 2A), whereas unphosphorylated double-headed myosin did not move at a significant rate (Fig. 2B). Single-headed myosin did not show this phosphorylation-dependent regulation of actin filament movement (Fig. 2, C and D). The mean velocities in µm/s of the actin filaments that were consistently moving (i.e. where the ratio of the standard deviation to the mean is less than 0.3) (23) were 1.5 ± 0.3 for A, 0.13 ± 0.12 for B, 0.95 ± 0.31 for C, and 0.65 ± 0.24 for D. The mean rate of movement for single-headed myosin with tpRLC was similar to the mean rate for unphosphorylated single-head myosin. The percentage of actin filaments that were moving was high in the case of single-headed myosin regardless of thiophosphorylation (Fig. 2, C and D). These data suggest that two heads are required for phosphorylation-dependent regulation of actin filament movement.

Figure 2: In vitro motility assays of myosins. All samples were exchanged with indicated RLC and treated as described for the samples in Table 1. The histograms show the velocity of all actin filaments observed in the field of view. A, thiophosphorylated double-headed myosin; B, unphosphorylated double-headed myosin; C, thiophosphorylated single-headed myosin; D, unphosphorylated single-headed myosin. The blackbars represent the velocities that are indistinguishable from the apparent velocity of filaments that are not moving (i.e. filaments bound to myosin heads in the absence of ATP(40) ). For A, these data were similar to data collected for samples that were phosphorylated during the motility assay on the coverslip.

The single-headed myosin could lack regulation because it contains papain-cleaved heavy chains (Fig. 1B), even though double-headed species with similarly cleaved chains by Staphylococcus aureus protease(7) , chymotrypsin(6) , or myopathic hamster protease (28) are known to be well regulated. To address this point, we prepared 95% purified single-headed gizzard myosin (data not shown) by digesting with S. aureus protease (15) . Neither the S.aureus- nor the papain-prepared single-headed myosins showed evidence for a subpopulation of regulated molecules in single turnover experiments (29) using formycin triphosphate. ()Thus, the unregulated nature of single-headed myosin is most readily attributed to the absence of one head.

These data show that an interaction between the two heads, rather than interaction of the head with the tail, is critical for the ``off'' state of unphosphorylated smooth muscle myosin. Phosphorylation at Ser-19 of the RLC relieves this inhibitory interaction, allowing the myosin to adopt force-generating conformations in the presence of actin. The lack of head-head interaction can explain why isolated myosin heads (S1) are active in both the phosphorylated and unphosphorylated states(7, 8) . Furthermore, as expected for molecules capable of head-head interaction, all double-headed proteolytic fragments studied to date have been found to be regulated(6, 7, 30) .

By analogy with the three-dimensional structure of skeletal myosin heads (31) and a fragment of the scallop myosin head(14) , Ser-19 of the smooth muscle myosin RLC is within a disordered amino terminus that may bind to portions of the myosin structure that were not visualized in the two crystal structures(31) . This binding region could be the coiled-coil S2 region or the other head, via either the heavy chain or the light chain regions. Phosphorylation probably effects the conformation of the amino terminus of the RLC as phosphorylation protects against proteolysis(32) . Perhaps upon phosphorylation of the RLC within a myosin molecule, elements of this amino-terminal portion of the light chain destabilize the interaction between the heads, probably by affecting the conformation at the hinge region (33) between the head and the tail. Residues 13-16 of the RLC of gizzard myosin appear to fit the requirements of such an element. Without these residues, the actin-activated ATPase remains inhibited regardless of phosphorylation. Furthermore, regulated and unregulated vertebrate myosin RLCs do not share homology in this region(34) . Although the exact mechanism of action of this amino-terminal portion of the light chain is not clear, the COOH-terminal portion of the RLC is likely to be an essential element(27) .

It has been shown that phosphorylation of both heads must occur before the actomyosin ATPase is fully activated(35, 36, 37) . In addition, dephosphorylation of one head appears to be sufficient to deactivate the whole molecule in both the filamentous and monomeric states(37) . In light of the importance of head-head interaction in the unphosphorylated state, it appears that in singly phosphorylated myosin, the phosphorylated head remains inactive because the other unphosphorylated head still retains elements of interaction with the phosphorylated head, thus restricting it from adopting force-generating conformations in the presence of actin. Upon double phosphorylation, both heads would become free, and the entire molecule could adopt the fully active conformation.

When bound to actin in the absence of ATP, the two heads of unphosphorylated smooth muscle heavy meromyosin interact asymmetrically between Lys-65, within a -barrel extending away from the bulk of the head(38) , and Glu-168 on the opposite side of the head(38, 39) . Interestingly, the amino acids corresponding to the -barrel are missing from unconventional myosin Is, which are single-headed and do not form filaments. This -barrel may be a critical domain, which is involved in the intramolecular head-head interactions that we propose are regulated by phosphorylation in smooth muscle myosin.

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.: 509-335-2428 or 509-335-2533; Fax: 509-335-9688.

()

The abbreviations used are: RLC, regulatory light chain; tpRLC, thiophosphorylated regulatory light chain; S1, subfragment 1 of myosin; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; ATPS, adenosine 5`-O-(thiotriphosphate).

()

C. R. Cremo, J. R. Sellers, and K. C. Facemyer, manuscript in preparation.

ACKNOWLEDGEMENTS

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				A. S. Rovner A Long, Weakly Charged Actin-binding Loop Is Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin J. Biol. Chem., October 23, 1998; 273(43): 27939 - 27944. [Abstract] [Full Text] [PDF]

				K. M. Trybus, V. Naroditskaya, and H. L. Sweeney The Light Chain-binding Domain of the Smooth Muscle Myosin Heavy Chain Is Not the Only Determinant of Regulation J. Biol. Chem., July 17, 1998; 273(29): 18423 - 18428. [Abstract] [Full Text] [PDF]

				L. D. Saraswat and S. Lowey Subunit Interactions within an Expressed Regulatory Domain of Chicken Skeletal Myosin. LOCATION OF THE NH2 TERMINUS OF THE REGULATORY LIGHT CHAIN BY FLUORESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., July 10, 1998; 273(28): 17671 - 17679. [Abstract] [Full Text] [PDF]

				M. Ikebe, T. Kambara, W. F. Stafford, M. Sata, E. Katayama, and R. Ikebe A Hinge at the Central Helix of the Regulatory Light Chain of Myosin Is Critical for Phosphorylation-dependent Regulation of Smooth Muscle Myosin Motor Activity J. Biol. Chem., July 10, 1998; 273(28): 17702 - 17707. [Abstract] [Full Text] [PDF]

				K. M. Trybus, Y. Freyzon, L. Z. Faust, and H. L. Sweeney Spare the rod, spoil the regulation: Necessity for a myosin rod PNAS, January 7, 1997; 94(1): 48 - 52. [Abstract] [Full Text] [PDF]

				M. Sata, W. F. Stafford III, K. Mabuchi, and M. Ikebe The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively PNAS, January 7, 1997; 94(1): 91 - 96. [Abstract] [Full Text] [PDF]

				V. N. Kalabokis, P. Vibert, M. L. York, and A. G. Szent-Gyorgyi Single-headed Scallop Myosin and Regulation J. Biol. Chem., October 25, 1996; 271(43): 26779 - 26782. [Abstract] [Full Text] [PDF]

				J. J. Olney, J. R. Sellers, and C. R. Cremo Structure and Function of the 10S Conformation of Smooth Muscle Myosin J. Biol. Chem., August 23, 1996; 271(34): 20375 - 20384. [Abstract] [Full Text] [PDF]

				K. Homma, J. Saito, R. Ikebe, and M. Ikebe Ca2+-dependent Regulation of the Motor Activity of Myosin V J. Biol. Chem., October 27, 2000; 275(44): 34766 - 34771. [Abstract] [Full Text] [PDF]

				H. L. Sweeney, L.-Q. Chen, and K. M. Trybus Regulation of Asymmetric Smooth Muscle Myosin II Molecules J. Biol. Chem., December 22, 2000; 275(52): 41273 - 41277. [Abstract] [Full Text] [PDF]