Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

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.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Ca2+-activated ATPase reaction of myosin from human cardiac, skeletal and smooth muscle cells

A comparative study of Ca2+ activated ATPase reaction of myosin isolated from cardiac, skeletal and smooth human muscles shows that on the molecules of all kinds of myosins there exist two types of centres binding the substrate CaATP with high and low affinity (Km approximately 1.6 x 10(-6) M and Km approximately 200 x 10(6) M respectively). Hydrolysis of ATP by myosin is noncompetitively activated by three types of Ca2+ binding sites on its molecule, having different affinity. The association constants (KaCa) for the smooth muscles are several times higher than those for skeletal and cardiac muscles.     

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

Early phosphate burst of heart myosins- Phylogenic variations.

The phosphate burst of cardiac myosins from different animal species was studied by a rapid quench-flow technique. The rate constant of the phosphate burst varied from one species to another. Moreover it was lower for cardiac myosins than for skeletal myosin. The phosphate burst rate correlated with the ATPase in the steady state and with the muscle-shortening speed. By contrast, the amplitude of the phosphate burst did not vary significantly. Its value was 0.8--0.9 per myosin site for each of the myosins studied. Deviations from this stoichiometry were related to the mode of preparation of the myosin and to denaturation by a high pH.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the actin-activated ATPase of aorta smooth muscle myosin

Phosphorylation of the 20,000-Da light chains, LC20, of vertebrate smooth muscle myosins is thought to be the primary mechanism for regulating the actin-activated ATPase activities of these myosins and consequently smooth muscle contraction. While actin stimulates the MgATPase activities of phosphorylated smooth muscle myosins, it is generally believed that the MgATPase activities of the unphosphorylated myosins are not stimulated by actin. However, under conditions where both unphosphorylated (5% phosphorylated LC20) and phosphorylated calf aorta myosins are mostly filamentous, the maximum rate, Vmax, of the actin-activated ATPase of the unphosphorylated myosin is one-half that of the phosphorylated myosin. While LC20 phosphorylation causes only a modest increase in Vmax, in the presence of tropomyosin, this phosphorylation does cause up to a 10-fold decrease in Kapp, the actin concentration required to achieve 1/2 Vmax. In the presence of low concentrations of tropomyosin/actin, a linear relationship is obtained between the fraction of LC20 phosphorylated and stimulation of the actin-activated ATPase. The relatively high actin-activated ATPase activity of unphosphorylated aorta myosin suggests that other proteins may be involved in the regulation of smooth muscle contraction. In contrast to the results presented here for aorta myosin, it has been reported that actin does not activate the MgATPase activity of unphosphorylated gizzard myosin and that the actin-activated ATPase of gizzard myosin increases more slowly than LC20 phosphorylation.     

Homologous calcium-binding proteins in the activation of skeletal, cardiac, and smooth muscle myosin light chain kinases

In order to identify the physiological regulator of calcium dependent myosin light chain kinases of cardiac, skeletal, and smooth muscles, the effects of the three homologous calciproteins, calmodulin, troponin C, and parvalbumin, on the kinases isolated from bovine myocardium, rabbit skeletal muscle, and turkey gizzard were examined. Only calmodulin was effective in stimulating the cardiac, skeletal, or smooth muscle kinase; troponin C and parvalbumin exhibited no activation of any of the three kinases, even when examined at concentrations as high as 10-(5) M. It is concluded that calmodulin is the specific regulator of myosin light chain kinase in cardiac, skeletal, and smooth muscle.     

Myosin-linked calcium regulation in vertebrate smooth muscle.

By the use of a new procedure, actomyosin may be extracted in high yield and purity from fowl gizzard which exhibits a calcium-dependent actin-activated ATPase activity comparable to that of the parent myofibril-like preparation. Studies of this vertebrate smooth muscle actomyosin show that the regulation of the actin-myosin interaction is effected, as in molluscan muscles, by the myosin molecule itself and not by an actin-linked regulatory system, as found in vertebrate skeletal muscle.Thus, calcium-sensitive smooth muscle actomyosin is composed of only myosin, actin and tropomyosin, any troponin-like components being absent. Myosin is the only component that binds significant amounts of calcium and shows a calcium-dependent actin-activated ATPase activity in the presence of F-actin from either gizzard or rabbit skeletal muscle.The cross-reaction of gizzard thin filaments with skeletal muscle myosin produces an actomyosin whose actin-activated ATPase is calcium-insensitive, showing that smooth muscle thin filaments do not serve a regulatory function.The effect of Mg²⁺ and pH, and evidence for the involvement of one of the myosin light chains in calcium regulation are described and discussed.     

A comparative study of reactive--SH groups of cardiac and skeletal muscle myosins.

Cardiac and skeletal muscle myosins have been treated by N-ethylmaleimide in presence or absence of Mg-ADP. The variations of Ca2+ and K+-ATPase activities and the incorporation of N-[14C]ethylmaleimide into the whole myosin molecule and into its separated subunits (heavy and light chains) have been measured with N-ethylmaleimide treatment for different lengths of time. The results reported here show the following: 1. The Ca2+-ATPase activity of cardiac myosin is activated by N-ethylmaleimide treatment to a lesser extent than that of skeletal myosin. 2. The K+-ATPase activity of both myosins is inhibited in the same quantitative way. 3. The cardiac light chain L1 contains one highly reactive thiol group which is absent from the skeletal light chains. 4. The labelling of three SH-groups localized in the heavy subunits of both myosins induced the same degree of inactivation. 5. The difference observed between the degree of inhibition of the Ca2+-ATPase activity for the two types of myosin with longer treatments appears to be due to differences in the reactivity of the fourth--SH group labelled on the heavy chains.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

Phosphorylation of the light-chain components of myosin from cardiac and red skeletal muscles.

1. The light-chain components of myosin from cardiac muscle (19000 and 27000 daltons) and of rabbit soleus and crureus muscles (19000, 27000 and 29000 daltons) were characterized. 2. The 19000-dalton components in carciac- and red-skeletal-muscle myosins were spontaneously modified to a component of slightly higher net negative charge. 3. The 19000-dalton component in cardiac and red skeletal muscles and their modified forms were phosphorylated by myosin light-chain kinase. 4. Evidence was obtained for the presence of myosin light-chain kinase in cardiac and red skeletal muscles. 5. Myosin light-chain kinase catalysed the phosphorylation of the whole light-chain fraction from white and red skeletal muscle at similar rates. The light-chain fraction of cardiac-muscle myosin was phosphorylated at a significantly lower rate. 6. The light-chain components of cardiac-muscle myosin and their phosphorylated froms were separated by ion-exchange chromatography and their amino acid compositions determined.     

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.     

Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cells

Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II-based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head-head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head-head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the 'blocked' myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head-head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II-based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.     

Distribution of isoelectric variants of the 17,000-dalton myosin light chain in mammalian smooth muscle

Isoelectric focusing of purified vascular smooth muscle myosin revealed two variants of the 17,000-dalton light chain subunits. The isoelectric points of the light chain variants were determined to be 4.13 (LC17a) and 4.19 (LC17b). Tryptic peptide maps of the two species of light chain generated by reverse-phase high performance liquid chromatography disclosed small but obvious differences in peptide composition while amino acid analyses of the variants were quite similar. Two-dimensional electrophoresis of extracts from various mammalian smooth muscles revealed tissue-specific differences in the relative content of LC17a and LC17b. Vascular (aorta, carotid, and pulmonary artery) muscles and tracheal smooth muscle contained both light chain variants while smooth muscle of the gastrointestinal tract (stomach and jejunum) contained LC17a only. The actin-activated Mg2+-ATPase activities of both phosphorylated and nonphosphorylated stomach (LC17b = 0) and aortic (LC17b = 40%) myosins were compared. In the presence of saturating tropomyosin, a 2-fold difference in Vmax was measured: phosphorylated, aortic, 0.119 +/- 0.009 versus stomach, 0.239 +/- 0.012 mumol of PO4 liberated/min/mg of myosin; nonphosphorylated, aortic, 0.065 +/- 0.004 versus stomach, 0.123 +/- 0.004 mumol of PO4 liberated/min/mg of myosin. In addition, the Vmax of myosin subfragment-1 ATPase from bovine aortic, pulmonary artery, and stomach myosins (LC17b contents, 40, 20, and 0%, respectively) was found to decrease in direct proportion to the LC17b content. Our results suggest that isoforms of the 17,000-dalton light chain subunits of mammalian smooth muscle myosin could play an important role in modulating actomyosin ATPase activity.     

Neural regulation of mammalian fast and slow muscle myosins: an electrophoretic analysis.

Mammalian nerves to fast and slow muscles have the remarkable property of changing the speed of contraction of muscles following cross-reinnervation. The biochemical basis of speed transformation is the change in myosin in ATPase activity. This paper provides electrophoretic evidence for structural changes in myosin from cross-reinnervated muscles. A method is described for the separation of intact fast and slow muscle myosins by polyacrylamide gel electrophoresis. This method utilizes the fact that ATP and its analogs prevent the formation of myosin polymers in low ionic strength buffers. In this system, normal fast muscle myosin has a higher electrophoretic mobility than slow muscle myosin. Normal rat soleus myosin has a major slow and a minor fast component due to two populations of muscle fibers. The same muscle cross-reinnervated by a fast muscle nerve shows only the fast component, The normal, homogeneous fast extensor digitorum longus muscle has only the electrophoretically fast myosin, but following cross-reinnervation it shows both fast and slow components. These results suggest that mammalian motor nerves can induce or suppress the expression of genes that code for fast and slow skeletal muscle myosins.