Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment-1

Myosin subfragment-1 from rabbit skeletal muscle was digested by thermolysin at 25 degrees, 12 degrees and 0 degree C. Thermolysin cleaves subfragment-1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N-terminus [Applegate, D. & Reisler, E. (1983) Proc. Natl Acad. Sci. USA 80, 7109-7112]. The rate of digestion at 25 degrees C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12 degrees C and completely eliminated at 0 degrees C. The results can be explained by assuming that there are two subfragment-1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004-2012], and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment-1 at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment-1. Actin inhibition of thermolysin digestion also prevailed when subfragment-1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment-1 modified by 4-phenylenedimaleimide [corrected], which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin-susceptible site of subfragment-1 at the 28-70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states.     

19F nuclear magnetic resonance as a probe of structural transitions and cooperative interactions in heavy meromyosin

An 19F NMR probe has been attached to the reactive sulfhydryl SH1 of the globular heads of rabbit skeletal heavy meromyosin. It serves as a sensitive monitor of the conformational state of the heads of heavy meromyosin in a manner similar to that seen for subfragment-1 (Shriver, J.W., and Sykes, B.D. (1982) Biochemistry 21, 3022-3028; Tollemar, U., Cunningham, K., and Shriver, J.W. (1986) Biochim. Biophys. Acta 873, 243-251). The NMR spectra indicate that there are at least two states for the heads in the SH1 region. The energetics of the interconversion of the two states of heavy meromyosin (HMM) differs significantly from that of S-1. In HMM in the absence of divalent cations, there are two reversible paths between the low temperature and high temperature states with a hysteresis-like behavior. One path is consistent with the head groups behaving independently and similar to S-1 alone. The second path indicates a coupling of the globular head region observed in S-1 with a second region forming a distinctly different cooperative unit. Upon addition of Ca(II) the hysteresis effect is lost and only the second cooperative unit is observed. Two explanations are offered for these results: the globular heads in HMM may couple with the S-2 segment, or the two globular heads of HMM may couple to form a larger cooperative unit. The ability to stabilize the larger cooperative unit with a divalent metal ion implicates a role for the LC2 light chain in coupling regions of the myosin molecule.     

A possible solvent effect of adenosine diphosphate influences the binding of 1,N6 ethenoadenosine diphosphate to myosin from skeletal muscle

Skeletal muscle myosin displays two independent and equivalent binding sites for 1,N6 ethenoadenosine diphosphate, with a dissociation constant of 24.7 microM. MgADP, 10 to 40 microM, behaves as a pure competitive type inhibitor (K(SI)=8-9 microM) for the binding of 1,N6 ethenoadenosine diphosphate to skeletal muscle myosin. On the contrary, the inhibition by MgADP, 0.11-1.54 mM, is neither competitive nor non-competitive nor mixed, as is revealed by the analysis with the general kinetic equation (K.J. Laidler, P.S. Bunting, The Chemical Kinetics of Enzyme Action, 2nd ed., Clarendon, Oxford, 1973, p. 94). To explain our finding we propose that MgADP operates a complex type of inhibition, acting both directly as a competitor for myosin active sites, and indirectly by perturbing the regions of the solvent near to the protein.     

Ligand-induced myosin subfragment 1 global conformational change

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.     

Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. We determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [14C]iodoacetamide or enzymatic activity measurements. We estimated the distribution of the IANBD on the fiber proteins to be approximately 77% on SH2, approximately 5% on SH1, and approximately 18% on troponin I. We characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization [Ajtai, K., & Burghardt, T. P. (1987) Biochemistry 26, 4517-4523]. With wavelength-dependent fluorescence polarization we use the ability to rotate the transition dipole in the molecular frame using excitation wavelength variation to investigate the three angular degrees of freedom of the cross-bridge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity and orientation of (iodoacetamido)proxyl spin-labeled myosin subfragment 1 decorating muscle fibers: localization of protein-bound spin labels using SDS-PAGE

The nitroxide spin label (iodoacetamido)proxyl (IPSL) was specifically and rigidly attached to sulfhydryl 1 (SH1) on myosin subfragment 1 (S1). The specificity of this label for SH1 was demonstrated by using a technique where the spin label is localized on the electrophoresis-isolated proteolytic fragments of myosin using electron paramagnetic resonance (EPR). Studies of the rigidity of the probe on SH1 indicate that the IPSL is immobilized on the surface of S1 in the presence and absence of the nucleotides MgADP or MgATP. The EPR spectrum of muscle fibers decorated with IPSL-S1 shows that the IPSL-S1 rotates from its orientation in rigor upon binding MgADP. The angular displacement due to nucleotide binding is larger than that detected with the (maleimido)tempo spin label [Ajtai, K., French, A. R., & Burghardt, T. P. (1989) Biophys. J. 56, 535-541], demonstrating that the IPSL is oriented on the myosin cross-bridge in a manner that is favorable for detecting cross-bridge rotation during the rigor to MgADP state transition.     

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.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase: III. Two ADP binding sites and their Mg(II) affinity; effects of vanadate and arsenate on enzymic complexes with ADP and 3-P-glycerate

31P nuclear magnetic resonance (NMR) measurements (at 121.5 MHz and 5 degrees C) were made on complexes of 3-phosphoglycerate kinase with ADP and 3-P-glycerate. Addition of Mg(II) to E.ADP shifts the alpha-P signal downfield by 3.8 ppm such that the alpha-P signal superimposes that for beta-P(E.MgADP). Such a shift is atypical among the Mg(II)-nucleotide complexes with other ATP-utilizing enzymes. This shift allowed the determination that enzyme bound ADP is saturated with Mg(II) for [Mg(II)]/[ADP] = 3.0--similar to that reported for ATP complexes with this enzyme (B.D. Ray and B.D. Nageswara Rao, Biochemistry 27, 5574 (1988]. This parallel behavior suggests that ADP binds at two sites on the enzyme as does ATP with disparate Mg(II) affinities. 31P relaxation times in E.MnADP.vanadate.3-P-glycerate and E.CoADP.vanadate.3-P-glycerate complexes indicate that these are long-lived, tightly bound complexes. 31P chemical shift measurements on diamagnetic complexes (with Mg(II] revealed three signals in the 2-5 ppm region (attributable to 3-P-glycerate) only upon addition of all the components necessary to form the E.MgADP.vanadate.3-P-glycerate complex. Subsequent sequestration of Mg(II) from the complex with excess EDTA reversed the Mg(II) induced effects on the ADP signals but did not cause coalescence of the three signals seen in the 2-5 ppm region. Addition of excess sulfate to dissociate these complexes from the enzyme resulted in a single resonance of 3-P-glycerate. The use of arsenate in place of vanadate yielded very similar results. These results suggest that, in the presence of MgADP, vanadate or arsenate, and 3-P-glycerate, the enzyme catalyzed the formation of multiple structurally distinguishable complexes that are stable on the enzyme and labile off the enzyme.     

Electrostatic contributions to the binding of myosin and myosin-MgADP to F-actin in solution

The ionic strength dependence of skeletal myosin subfragment 1 (S1) binding to unregulated F-actin was measured in solutions containing from 0 to 0.50 M added lithium acetate (LiOAc) in the absence and presence of MgADP. The data were analyzed by using a theory based on an ion interaction model that is rigorous for high ionic strength solutions [Pitzer, K. S. (1973) J. Phys. Chem. 77, 268-277] in order to obtain values for K, the equilibrium association constant when the ionic strength is zero, and for [zMzA[, the absolute value of the product of the net electric charges of the actin binding site on myosin (zM) and the myosin binding site on actin (zA). The presence of MgADP reduced K by a factor of 10, as expected, and reduced [zMzA[ by about 1 esu2. Because the presence of MgADP is not likely to change the net charge of the myosin binding site on actin, these data are consistent with a model in which MgADP binding to S1 reduces its affinity for actin by a mechanism that reduces the net electric charge of the acting binding site on S1. The value of [zMzA[ in the absence of ADP was 8.1 +/- 0.9 esu2, which, if one uses integer values, suggests that zM and zA are in the 8+ to 1+ esu and 1- to 8- esu ranges, respectively. ADP binding then reduces zM to the 7+ to 0.88+ esu range.     

Nucleotide- and temperature-induced changes in myosin subfragment-1 structure.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.     

Probing cross-bridge angular transitions using multiple extrinsic reporter groups.

15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic changes at the actomyosin-subfragment 1 interface during force-generating reactions.

The ionic strength dependence of the binding of rabbit skeletal muscle myosin subfragment 1, S1, to F-actin in the presence of saturating concentrations of MgATP or MgADP was analyzed in order to determine the association constants at zero ionic strength [K(0)] and the products of the net effective electric charges (magnitude of zMzA) at the binding interfaces. K(0) and magnitude of zM A were 1 x 10(6) M-1 and 17 esu2 for S1-MgADP,P, and 5 x 10(7) M-1 and 7 esu2 for S1-MgADP, respectively, for binding to F-actin at 25 degrees C. At ionic strengths near physiological, the increase in affinity is close to 10(4)-fold for this transition that may correspond to force generation in muscle fibers. The large, from 17 to 7 esu2, decrease in the electrostatic contribution to binding appears to be correlated with a much larger increase in nonelectrostatic interactions, unlike the simpler transition of actin-bound S1-MgADP to S1, which appears to be due entirely to electrostatic changes [Highsmith, S. (1990) Biochemistry 29, 10690-10694]. These results for acto-S1-MgADP,P to acto-S1-MgADP suggest that a substantial transformation of the actin binding site on S1 occurs even if there is a translocation to a new interface.     

Two-state equilibria of myosin subfragment 1 and its complexes with ADP and actin

We previously reported that the nucleotide complex of myosin subfragment 1, S1.epsilon ADP, exists in two states on the basis of the temperature dependence of the fluorescence decay of bound 1,N6-ethenoadenosine diphosphate (epsilon ADP) [Aguirre, R., Lin. S.-H., Gonsoulin, F., Wang, C.-K., & Cheung, H.C. (1989) Biochemistry 28, 799-809]. We have extended the previous study of the equilibrium between the two states, S1L.ADP in equilibrium S1H.ADP, by using a fluorescently labeled myosin S1 (S1-AF). In S1 alkylated with IAF [5-(iodoacetamido)fluorescein], the decay of the label emission was biexponential both in the presence and absence of ADP and/or actin. In the presence of ADP, the two decay times were 4.30 (alpha 1 = 0.55) and 0.80 ns (alpha 2 = 0.45) at 12.4 degrees C, in a medium containing 60 mM KCl, 30 mM TES (pH 7.5), and 2 mM MgCl2. The steady-state fluorescence intensities of S1-AF, (S1-AF).ADP, acto.(S1-AF), and acto.(S1-AF).ADP were dependent on temperature over the range of 5-30 degrees C. By combining lifetime and steady-state intensity data, we obtained for the two-state transition (S1-AF)L.ADP in equilibrium (S1-AF)H.ADP the following parameters: delta H degrees = 16.1 kcal/mol (67.3 kJ/mol) and delta S degrees = 55.8 cal/(deg.mol) [233.5 J/(deg.mol)], in agreement with previous results obtained with epsilon ADP. The delta H degrees values for the two-state transition of S1-AF, acto.(S1-AF), and acto.(S1-AF).ADP are 13.0, 21.6, and 5.2 kcal/mol, respectively. The corresponding delta S degrees values are 46.9, 79.5, and 17.4 cal/(deg.mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of actin to myosin subfragment 1 in the presence of nucleotides

Chemical cross-linking of actin to the 20K and 50K fragments of tryptically cleaved myosin subfragment 1 (S-1) by the zero-length cross-linking reagent 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC) was used as a probe of the acto-S-1 interface in the presence of nucleotides. The course of the two reactions was monitored by measuring on sodium dodecyl sulfate (SDS)-polyacrylamide gels the time-dependent formation of the 20K-actin and 50K-actin cross-linked products. Both reactions were inhibited somewhat in the presence of MgADP, were slowed 3-4-fold in the presence of magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and proceeded at least 7-fold slower with N,N'-p-phenylenedimaleimide (pPDM) modified S-1, as compared to the respective rates in the absence of nucleotides. However, neither the binding of the nucleotides MgADP and MgAMPPNP to S-1 nor the modification of S-1 by pPDM significantly changed the ratio of the cross-linking rates of actin to the 20K and 50K fragments. Similar to what was previously observed in the absence of nucleotides [Chen, T., Applegate, D., & Reisler, E. (1985) Biochemistry 24, 137-144], actin was cross-linked at an approximately 3-fold faster rate to the 20K fragment than to the 50K fragment under all reaction conditions tested. Thus, irrespective of the extent of acto-S-1 dissociation or the binding of nucleotides to acto-S-1, the 20K fragment remains the preferred cross-linking site for actin. These results show that the interaction of actin with each of the cross-linking sites on S-1 is not under selective or preferential control by nucleotides.     

Elementary steps of the cross-bridge cycle in fast-twitch fiber types from rabbit skeletal muscles

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca(2+)-activated at 20 degrees C and the effects of MgATP, phosphate (P, P(i)), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme: where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k(2), 198-526 s(-1); k(-2), 51-328 s(-1); k(4), 13.6-143 s(-1); k(-4), 13.6-81 s(-1)) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k(-2), k(4)) varied more among fiber types than their reversals (k(2), k(-4)). The equilibrium constants K(1) (MgATP affinity) and K(2) (=k(2)/k(-2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K(4) (=k(4)/k(-4), force generation) and K(5) (P(i) affinity) were larger in IIB than IIA and IID fibers. K(1) showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM(-1)), IID (4.9 mM(-1)), and IIB (0.84 mM(-1)). Similarly, the MgADP affinity (K(0)) was larger in type IID fibers than in type IIB fibers.     

Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.

We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.     

Actin and temperature effects on the cross-linking of the SH1-SH2 helix in myosin subfragment 1

Past biochemical work on myosin subfragment 1 (S1) has shown that the bent alpha-helix containing the reactive thiols SH1 (Cys(707)) and SH2 (Cys(697)) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5-15 A in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25 degrees C, the rate constants for cross-linking in S1 alone are low, with values of approximately 0.010 min(-1) for all of the reagents. At 4 degrees C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min(-1). The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4 degrees C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.     

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.