Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Evidence for heavy chain-alkali light chain association-dissociation equilibrium

Modification of the free alkali light chains of myosin by iodoacetylation results in a much lower extent of exchange into myosin subfragment 1 by the thermal hybridization procedure (Burke, M., and Sivaramakrishnan, M. (1981) Biochemistry 20, 5908-5913). As reported by others (Wagner, P. D., and Stone, D. B. (1983) J. Biol. Chem. 258, 8876-8882), free alkali light chains modified by iodoacetate at their single sulfhydryl residue exhibit minimal exchange into intact myosin. However, when unmodified alkali light chain is used to probe for exchange, close to the theoretical limit of exchange is observed for subfragment 1, and significant levels of exchange are found for myosin. It appears that modification of the free alkali light chain alters the structure of the protein, and this causes either a marked reduction in its affinity for the heavy chain or in its ability to enter the light chain binding site. This conclusion is supported by tryptic digestions done on the unmodified and modified free light chains where it is found that the latter is degraded at a much faster rate, indicating a more open structure for the modified protein. The observation that alkali light chain exchanges into myosin when unmodified alkali light chains are used indicates that the presence of the associated 5,5'-dithiobis-(2-nitrobenzoic acid) light chains does not preclude the reversible dissociation of this subunit from myosin under ionic and temperature conditions approaching the physiological state.     

Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.

The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids

The formation of hybrid myosin and subfragment 1 species by incubation of these proteins with free alkali light chains at physiological ionic and temperature conditions is described. Exchange of bound alkali light chain on myosin by free alkali light chains under these conditions is readily demonstrated from the subunit composition of the isolated myosin. Therefore, the light chain exchange previously described for the one-headed subfragment 1 [Sivaramakrishnan, M., & Burke, M (1981) J. Biol. Chem. 256, 2607--2610] also occurs in the two-headed myosin molecule. It is found than the isozyme to hybrid transformation is dependent on both the temperature and the ionic strength of the incubation mixture but is relatively independent of pH in the range 6.5--8.0. A comparison of the SF1(A1) leads to SF1(A2)h system with the SF1(A2) leads to SF1(A1)h system indicates that more hybrid is formed in the latter case. With the assumption that hybrid formation reflects the degree of reversible dissociation exhibited by the isozyme, under the particular experimental condition employed, the data signify that the subunit interactions in the two isozymes are not identical and that the heavy chain--A1 interactions are significantly more stable that the heavy chain--A2 ones. An examination of the ATPase properties of the thermal hybrids in the presence and absence of actin indicates close similarities to their corresponding "native" isozymic counterparts.     

Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature

Evidence is presented that under physiological conditions of ionic strength and temperature, where myosin Subfragment 1 is hydrolyzing MgATP, the interaction between its subunits is extremely labile. Incubation of [3H]N-ethylmaleimide-SF1(A1) with N-ethylmaleimide-SF1(A2) in the presence of 10 mM MgATP at 37 degrees C resulted in the exchange of subunits between these isozymes. This is readily discernible from the subunit composition and distribution of the 3H label after separation of the isozymes by ion exchange chromatography. Moreover, incubation of unmodified SF1(A1) or SF1(A2) with the free Alkali light chains A2 and A1, respectively, under the same conditions led to the formation of significant amounts of the hybrid species. These findings suggest that in vivo the Alkali light chain-heavy chain interaction of Subfragment 1 is in a state of dynamic equilibrium between associated and dissociated states.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Structural characteristics and aggregation of myosin heads in skeletal muscles

Native conformational modifications of rabbit skeletal muscle myosin and its subfragment-1 (S-1) within the temperature range of 0-40 degrees C and irreversible unfolding of these proteins structure at temperatures 40-70 degrees C have been studied by the fluorescence and light scattering methods. The results obtained permit stating that myosin and its active subfragments form associates at the concentrations above 0.3 microM. Hydrophobic interactions between definite sites of S-1 are likely to be primarily responsible for the association. The complex profile of S-1 melting curve at high ionic strength indicates the existence of three structural domains in the heavy chain of the myosin head.     

A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

Characterization and localization of myosin in the brush border of intestinal epithelial cells

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.     

Calponin binds to the 20-kilodalton regulatory light chain of myosin

Calponin (CaP) is a 34 kDa smooth muscle-specific protein that has been implicated in regulation of smooth muscle contractility. Two CaP binding sites on smooth muscle myosin rod have been recently described [Szymanski and Tao (1997) J.Biol.Chem. 272, 11142-11146]. We used a combination of cosedimentation, overlay, and fluorescence assays to determine the interaction between CaP and both subfragment 1 of myosin and isolated 20 kDa regulatory light chain of myosin (RLC). Subfragment 1, which was generated by cleavage of myosin with Staphylococcus aureus protease (myosin S1SA) inhibits cosedimentation of CaP with myosin filaments. Fluorescence assay showed that CaP labeled with fluorescent label (DAN-CaP) interacts with myosin S1SA in solution via a single class of binding sites. The binding constant (kaff) of this interaction at 50 mM NaCl is (2. 1 +/- 0.2) x 10(6) M-1 (n = 3). The interaction between DAN-CaP and myosin S1SA depends on ionic strength, and the EC50 of inhibition of this interaction occurs at about 130 mM NaCl. In contrast, the subfragment 1 that was generated by papain digestion (myosin S1PA), which cleaves RLC 4 kDa away from the NH2-terminal end of the molecule, does not interact with DAN-CaP. Overlay and fluorescent assay in solution showed that CaP binds to isolated RLC, suggesting that the interaction between CaP and subfragment 1 of myosin is due to a direct binding of CaP to RLC. CaP binding to myosin S1SA is stronger than to subfragment 2 in physiological salt concentrations. CaP binding to myosin head strengthened upon phosphorylation of RLC by Ca2+/calmodulin-dependent myosin light chain kinase. We suggest that CaP binds to subfragment 1 of myosin, exclusively via the NH2-terminal end of RLC, and this interaction could play a role in regulation of the actin-myosin interaction in smooth muscle contractility.     

Conformational transitions within the head and at the head-rod junction in smooth muscle myosin studied with a limited proteolysis method

It was previously shown that tryptic digestion of subfragment 1 (S1) of skeletal muscle myosins at 0 degree C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH2-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25 degrees C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH2-terminus is involved in the communication between the sites of nucleotide and actin binding. We also show here for the first time that the S1-S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain-produced S1 devoid of the regulatory light chain) is also temperature-dependent but insensitive to nucleotides and actin. It is suggested that the temperature-dependent alteration in the flexibility of the head-rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J. Mol. Biol. 182, 271-279; Redowicz, M.J. & Strzelecka-Go?aszewska, H. (1988) Eur. J. Biochem. 177, 615-624], may contribute to the temperature dependence of some steps in the cross-bridge cycle.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Effect of cations and temperature on kinetics of desmin assembly.

Smooth-muscle desmin, which was isolated from avian gizzard, was purified and used to form reconstituted intermediate filaments. Filament assembly was done in the presence of physiological cations, Ca2+, Mg2+, Na+, and Na+ plus Mg2+, and with non-physiological cations Cu2+ and Ni2+. Assembly was done at 2 degrees, 22 degrees and 37 degrees C, and was monitored by absorbance and by electron microscopy. Absorbance increased most rapidly during the first 2-5 min and then increased at a slower rate with the physiological cations, but decreased after that time with the non-physiological cations. For each physiological cation, absorbance increased with increasing temperature. This was particularly evident with Ca2+, which produced the lowest absorbance at 2 degrees C and the highest at 37 degrees C. When ionic strength was comparable, filament-forming buffers that contained bivalent cations were associated with higher absorbance values. Filament diameters were significantly smaller 60 min after assembly initiation than after 5 min. Average filament diameters, when formed in the presence of Cu2+ or Ni2+, were 10% greater than in the presence of the physiological cations and did not show a consistent tendency to decrease as time increased. These results demonstrate the importance, not only of the pH and ionic composition of the filament-forming buffer, but also of the temperature and duration of dialysis for reconstitution of desmin filaments.     

Kinetic characterization of a monomeric unconventional myosin V construct.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.     

Calcium sensitivity of vertebrate skeletal muscle myosin

The calcium sensitivity of vertebrate skeletal muscle myosin has been investigated. Adenosinetriphosphatase (ATPase) activity was assayed in a reconstituted system composed of either purified rabbit myosin plus actin or myosin plus actin, tropomyosin, and troponin. The calcium sensitivity of actomyosin Mg-ATPase activity was found to be directly affected by the ionic strength of the assay medium. Actomyosin assayed at approximately physiological ionic strength (120 mM KCl) demonstrated calcium sensitivity which varied between 6 and 52%, depending on the myosin preparation and the age of the myosin. Mg-ATPase activity was increased when calcium was present in the assay medium at physiological ionic strength. Conversely, actomyosin Mg-ATPase activity assayed at a lower ionic strength (15 mM KCl) was inhibited by addition of calcium. Addition of tropomyosin and troponin to the assay increased the calcium sensitivity of the system at the physiological ionic strength still further (up to 99% calcium sensitivity) and conferred calcium sensitivity on the system at the lower ionic strength (greater than 90% calcium sensitivity). A correlation also existed between myosin's calcium sensitivity and the phosphorylated state of light chain 2.     

Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro

We have reinvestigated the effects of Ca++ and ATP on brush borders isolated from intestinal epithelial cells. At 37 degrees C, Ca++ (1 microM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37 degrees C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++ (1 microM), and occurs in both membrane-intact and demembranated brush borders. Ca++ - dependent-solation of microvillus cores requires a concentration of Ca++ slightly greater (10 microM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC20), is stimulated by Ca++. At 37 degrees C, BBMLC20 phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20 phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.     

Substructure of skeletal myosin subfragment 1 revealed by thermal denaturation

The stability of myosin subfragment 1 (S1) to thermal denaturation has been followed by limited tryptic proteolysis. Digestions done during the thermal denaturation show that at temperatures at and above 37 degrees C there is a marked increase in the susceptibility of S1 to tryptic degradation, as evidenced by the loss of all bands corresponding to the normally trypsin-resistant fragments of 50, 27, and 21 kDa of the heavy chain and to the light chain. The enhanced digestion of S1 appears to be due to a general unfolding of all segments of S1, although the 50-kDa segment appears to unfold at a lower temperature than the remainder of the S1 structure. Digestions done after 30-min exposure to higher temperatures or after subsequent cooling to 25 degrees C show marked differences in the susceptibility of the S1 to trypsin. This suggests that, on cooling, a substantial portion of the S1, but not the 50-kDa segment, is capable of refolding to a state corresponding closely to that in the native S1. These data indicate that in terms of thermal denaturation the S1 behaves as though it is comprised of two domains--an unstable 50-kDa domain and a more stable domain comprised of the 27- and 21-kDa segments of the heavy chain interacting with the light chain, as proposed recently by Setton and Muhlrad [Setton, A., & Muhlrad, A. (1984) Arch. Biochem. Biophys. 235, 411-417]. The rates of thermal inactivation of the ATPase of S1 are found to correspond closely to the decay rates for the 50-kDa fragment, suggesting that this segment in S1 is closely associated with the ATPase function of the protein.     

Electric birefringence study of the solution structure of chymotrypsin-cleaved Acanthamoeba myosin II

After removal of the 66 COOH-terminal amino acids from each of its two heavy chains by chymotrypsin digestion, Acanthamoeba myosin II forms only parallel dimers under conditions in which native myosin II forms bipolar filaments (Kuznicki, J., Cote, G. P., Bowers, B., and Korn, E. D. (1985) J. Biol. Chem. 260, 1967-1972). We have studied the solution structure of the chymotrypsin-cleaved myosin II by electric birefringence. Only two species, known to be monomer and parallel dimer from previous studies, were detected. The contribution to the birefringence decay from dimer increased from about 10 to 70% as the KCl concentration was lowered from 100 mM to 0 in 50% glycerol. At all ionic strengths, the monomer had a relaxation time corrected to water at 20 degrees C of 8.2 microseconds, whereas a relaxation time of 10.3 microseconds was expected for monomers with straight rigid rods. This strongly indicates that the myosin rod in solution is bent. On the assumption that there is a single bend 26 nm from the tip of the tail, as suggested by electron microscopy, it was calculated that the average bend angle would be 110 degrees, in solution, if as seems most likely, the average angle between the two globular heads were 180 degrees. The observed relaxation time of the dimer corrected to water at 20 degrees C was 25 microseconds, independent of ionic strength, which, if the motion of the heads were unrestricted, is consistent with a structure for a parallel dimer in which either the two monomer subunits have straight rigid rods and are staggered by about 28 nm or only one is bent and the stagger is 30 nm. As described in the accompanying Appendix, either of these dimers can be assembled into a bipolar filament compatible with the apparent structure of filaments of native myosin II (Pollard, T.D. (1982) J. Cell Biol. 95, 816-825).     

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.