Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

The vitamin C transporter SVCT2 is down-regulated during postnatal development of slow skeletal muscles

Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

Rate-limiting step in the actomyosin adenosinetriphosphatase cycle: studies with myosin subfragment 1 cross-linked to actin

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

ELECTRON MICROSCOPE OBSERVATIONS ON MYOSIN FROM PHYSARUM POLYCEPHALUM

Myosin has been separated from Physarum polycephalum actomyosin in confirmation of the results of Hatano and Tazawa. In an intermediate step, myosin-enriched actomyosin has also been obtained. The mean yield of free myosin was 4.4 mg from 100 g of mold. It was obtained as water-clear solutions at µ = 0.055 with calcium ATPase activity of up to 0.5 µM P_i/min per mg. Negatively stained preparations were examined by electron microscopy. Physarum myosin in 0.5 M KCl interacted with actin from rabbit skeletal muscle to form polarized arrowhead complexes similar to but less regular than those of natural actomyosin from muscle or myosin-enriched Physarum actomyosin. The Physarum myosin-enriched actomyosin at low ionic strength displayed evidence of head-to-tail and tail-to-tail aggregation attributable to the myosin component. Yet Physarum myosin alone did not produce detectable filaments at µ = 0.055 at pH 7, 6.5, or 5.8, nor when dialyzed against 0.01 M ammonium acetate, nor when the dielectric constant of the medium was reduced. However, aggregation approaching the extent of ‘thick filaments’ up to 0.3 µ long was found in some preparations of myosin-enriched actomyosin put into solutions containing adenosine triphosphate. Myosin alone in such solutions did not form filaments. The results are compatible with the idea that head-to-tail aggregations are favored by actin-myosin interactions in Physarum, possibly due to alignment of the extended or tail portions of this myosin molecule.     

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.     

Evaluation of different bovine muscles to be applied in freeze-drying for instant meal. Study of physicochemical and senescence parameters

The aim of the present research was to evaluate bovine muscles to be subjected to freeze-drying for an instant meal. Physicochemical and senescence parameters were evaluated. The experimental part was divided into two steps. In the first step, the Semitendinosus muscle was chosen to establish methodology and experimental conditions. Physicochemical, microstructure and senescence parameters were analysed. In the second step, economic bovine muscles such as Semimembranous and Spinalis dorsi were subjected to the same methodology and conditions as in the first step in order to compare them by analysing the same parameters. L* and a* values were statistically significant (P<0.05) for Semimembranous and Spinalis dorsi muscles, showing differences among condition effects and in muscles. Humidity and water activity showed among the muscles analysed that cooked and rehydrated samples did not exhibit differences. Microstructure of Semitendinosus and Semimembranous were not separated and fragmented as occurred with Spinalis dorsi after freeze-drying. Results allowed us to select among the muscles studied that Semimembranous was suitable and economic to be used in an instant meal.     

Chronic stimulation of the spinal cord in developing chick embryo causes the differentiation of multiple clusters of acetylcholine receptor in the posterior latissimus dorsi muscle

Electrodes were implanted around the spinal cord of 7-day-old chick embryos and electric pulses delivered at 0.5-Hz frequency from the 10th to 15th day of incubation. At Day 15, the posterior latissimus dorsi (PLD) muscle, which, in control animals, is focally innervated, was dissected. The number and distribution of AChR clusters revealed by autoradiography after labeling with ¹²⁵I-α-bungarotoxin was quantitatively studied on isolated muscle fiber fragments and on serial sections of the whole muscle. After chronic stimulation, muscle fibers with multiple AChR clusters were observed. The distribution of the clusters appeared less regular than in the anterior latissimus dorsi muscle which, in control embryos, receives a multiple innervation. The total number of AChR clusters per PLD muscle increased about 1.8 times as a consequence of the stimulation without significant change of the total number of muscle fibers.     

Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus (K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.     

AMP and IMP Dissociate Actomyosin into Actin and Myosin

We investigated to determine why heating of squid muscle at 60 °C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 °C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 °C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Calponin-like protein from mussel smooth muscle is a competitive inhibitor of actomyosin ATPase

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V_max and K_ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K_ATPase value and to a lesser extent the decrease in V_max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V_max and K_ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.     

Potential phasic and tonic muscles express a common set of fast and slow myosin light chains and fast tropomyosin during early development of chick embryo

We investigated the expression of myosin light chains and tropomyosin subunits during chick embryonic development of the anterior (ALD) and posterior (PLD) parts of the latissimus dorsi muscles. As early as day 8 in ovo, both muscles accumulate a common set of myosin light chains (LC) in similar ratios (LC1F : 55 per cent; LC2S : 25 per cent; LC2F : 12 per cent ; LC1S : 8 per cent) and a common set of tropomyosin (TM) subunits (β², β¹, α²_F).Later during development, the slow components of the LC regularly disappear in the PLD and the fast components of the LC and the α²_FTM disappear in the ALD, so that the adult pattern is almost established at the time of hatching.Thus, early in development, the two muscles accumulate a common set of fast and slow myosin light chains and fast tropomyosin and some isoforms are repressed at a later stage during development. These data might suggest that during development, the regulatory mechanisms of muscle specific isoform expression differ from one contractile protein to another.     

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3 M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤ 1 M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3 M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, V_max, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3 M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3 M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.     

Increased trunk muscle recruitment during the golf swing is linked to developing lower back pain: A prospective longitudinal cohort study

BackgroundThis is the first study that presents electromyographic measurements prior to the development of lower back pain in young elite golfers.Study designProspective longitudinal cohort study.MethodsThirty-three injury free elite golfers were included. Muscle activity from latissimus dorsi, rectus abdominis, external oblique and erector spinae muscles were recorded during 10 drive golf swings. Lower back pain, training and performance were monitored over a six-month period. Muscle activation comparisons were made between the baseline results of those who went on to develop lower back pain versus those who did not go on to develop lower back pain.ResultsAfter the six-month monitoring period 17 participants developed lower back pain. The group that developed lower back pain had increased dominant rectus abdominis and dominant latissimus dorsi activation at various time points throughout the swing.DiscussionThe increased dominant rectus abdominis and dominant latissimus dorsi during the golf swing is linked with developing lower back pain. Training strategies aimed at reducing these muscles activation during the swing may reduce the incidence of lower back pain in young elite male golfers.     

Activation of skeletal S-1 ATPase activity by actin-tropomyosin-troponin. Effect of Ca++ on the fluorescence transient.

Regulation in striated muscles primarily involves the effect of changes in the free calcium concentration on the interaction of subfragment-1 (S-1) with the actin-tropomyosin-troponin complex (henceforth referred to as [acto]R). At low concentrations of free Ca++ the rate of ATP hydrolysis by (acto)R S-1 can be as much as 20-fold lower than that in the presence of high free Ca++, even though the binding of S-1 to (actin)R in the presence of ATP is virtually independent of the calcium concentration. This implies that the mechanism of regulation involves a kinetic transition between actin-bound states, rather than the result of changes in actin binding. In the current work, we have investigated the fluorescence transient that occurs with the binding and hydrolysis of ATP both at low and high free [Ca++]. The magnitude of this transition at low free [Ca++] is higher than at high free [Ca++]. At low free [Ca++], the rate of the fluorescence transient either stays constant or decreases slightly with increasing free actin concentrations, but at high free [Ca++] the rate increases slightly with increasing free actin concentration. The observed changes in rate are not great enough to be of regulatory importance. The results of the fluorescence transient experiments together with the binding studies performed at steady state also show that neither the binding of M.ATP or M.ADP.Pi to (actin)R is appreciably Ca++ sensitive. These data imply that an additional step (or steps) in the ATPase cycle, i.e., other than the burst transition, must be regulated by calcium.     

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl₂, HMM hydrolyzed 3PP at a maximum rate of 0.016 s⁻¹ HMM⁻¹, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.     

Differences between smooth and skeletal muscle myosins in their interactions with F-actin

Myosin and F-actin were prepared from bovine carotid arterial smooth muscle and the properties of the binding of myosin to F-actin were compared with those of the binding of skeletal muscle myosin to F-actin. The following differences were observed between skeletal and smooth muscle myosins. 1. The rate of ATP-induced dissociation of arterial actomyosin was equal to that of hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin, but was much lower than those of skeletal muscle actomyosin and of hybrid actomyosin reconstituted from skeletal muscle myosin and arterial F-actin. 2. The amount of ATP necessary for complete dissociation of arterial actomyosin was 2 mol/mol of myosin, although it is well known that skeletal muscle actomyosin is dissociated completely by the addition of 1 mol ATP per mol of myosin. 3. Arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin did not dissociate upon addition of 0.1 mM PPi, while skeletal muscle actomyosin dissociated completely. 4. In the absence of Mg2+, neither dissociation by ATP nor ATPase [EC 3.6.1.3] activity was observed with arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin. On the other hand, skeletal muscle actomyosin dissociated almost completely upon addition of ATP and showed a considerably high ATPase activity. These observations reveal marked differences between myosins from skeletal and smooth muscles in their binding properties to F-actin.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.