Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis

We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle.     

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.     

Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.     

Orientation of spin-labeled myosin heads in glycerinated muscle fibers.

We have used electron paramagnetic resonance (EPR) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas muscle fibers. Because the angle between the magnetic field and the principal axis of the probe determines the position of the EPR absorption line, spectra from labeled fibers oriented parallel to the magnetic field yielded directly the distribution of spin label orientations relative to the fiber axis. Two spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. In rigor fibers with complete filament overlap, both labels displayed a narrow angular distribution, full width at half maximum approximately 15 degrees, centered at angles of 68 degrees (IASL) and 82 degrees (MSL). Myosin subfragments (heavy meromyosin and subfragment-1) were labeled and allowed to diffuse into fibers. The resulting spectra showed the same sharp angular distribution that was found for the labeled fibers. Thus is appears that virtually all myosin heads in a rigor fiber have the same orientation relative to the fiber axis, and this orientation is determined by the actomyosin bond. Experiments with stretched fibers indicated that the spin labels on the fraction of heads not interacting with actin filaments had a broad angular distribution. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting in a spectrum almost indistinguishable from that of an isotropic distribution of probes. Addition of either an ATP analog (AMPPNP) or pyrophosphate produced partial disorder. That is a fraction of the probes remained sharply oriented as in rigor while a second fraction was in a disordered distribution similar to that of relaxed fibers.     

Three distinct actin-attached structural states of myosin in muscle fibers

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M'.D.P, we used pPDM to cross-link SH1 (Cys(707)) to SH2 (Cys(697)) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M'.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M'D.P analog on the force generation pathway.     

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.     

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray study of myosin heads in contracting frog skeletal muscle

Using synchrotron radiation, the behaviour of the diffuse X-ray scatter was investigated in the relaxed and active phases of auxotonic and isometric contractions. Muscles were stimulated tetanically for 0.75 of a second, leaving intervals of three minutes between successive contractions. In isometric contractions the scatter is very asymmetric, which means that the myosin heads have a strongly preferred orientation. During tension rise the scatter expands in the meridional direction and contracts in the equatorial direction, the maximal local intensity change being about 20%. The shape change indicates that on average the myosin heads become oriented more perpendicularly to the fibre axis. The distribution of orientations at peak tension is quite different from that we found previously in X-ray scattering data from rigor muscles. In auxotonic contractions where muscles shorten against an increasing tension the scatter is practically circularly symmetrical. This suggests that during shortening the myosin heads go evenly through a wide range of orientations. It is concluded that the results from both the auxotonic and isometric experiments provide strong support for the rotating myosin head model. In isometric contractions the transition between the relaxed phase and peak tension is accompanied by an overall increase in scattering intensity of about 10%: this corresponds to a relative increase in the fraction of disordered myosin heads by almost 30%.     

Different phosphorylated forms of myosin in contracting tracheal smooth muscle

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

An electron spin resonance study of temperature-induced structural changes of the spin-labeled myosin head: an evidence for two conformational states of myosin head

Heavy meromyosin labeled at the SH1 thiol group with an iodoacetamide spin label was studied by electron spin resonance spectroscopy at various temperatures in the presence and absence of nucleotides and PPi. The electron spin resonance spectra of the spin label bound to myosin head showed temperature-dependent changes indicating changes of the structure around the SH1 thiol group of the myosin head. As the temperature was elevated, the bound spin label was more mobilized in all the systems examined. The mobilization of the bound spin label by the elevation of temperature was enhanced in the presence of nucleotides or PPi. The temperature-dependent spectral changes had isosbestic points indicating that the structural changes around the SH1 thiol group took place between two states of the bound spin label, a weakly immobilized and a strongly immobilized state.     

Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.     

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Saturation transfer electron paramagnetic resonance.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78-88% of the heads in the mobile state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.