Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.     

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.     

Preparation and characterization of heavy meromyosin and subfragment 1 from vertebrate cytoplasmic myosins

The soluble fragments of myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) have been instrumental in elucidating the kinetic mechanisms of the actin-activated MgATPase activity of both skeletal and smooth muscle myosin. To date, relatively little has been published on these fragments from vertebrate cytoplasmic myosins. We now describe the preparation and steady-state kinetic characterization of S-1 and HMM from human platelet and avian intestinal epithelial brush border myosin. The HMM prepared from each of these tissues was similar both in their SDS-polyacrylamide gel pattern and in their steady-state kinetic properties. The Vmax of the actin-activated MgATPase activity varied between 0.8 and 2.5 s-1, and the KATPase (the apparent dissociation constant derived from a double-reciprocal plot of the MgATPase activity) was about 1-2 microM. This low value for the apparent dissociation constant was similar to the dissociation constant of HMM for actin directly measured under similar conditions and is about 40 times lower than that determined with avian smooth muscle HMM. The KATPase of the cytoplasmic HMM was only slightly increased when the ionic strength was raised from 12 to 112 mM.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.     

The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state

The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction.     

Myosin I: A new insight into the mechanism and cellular significance of actin-based motility

From our work on brush border myosin I structure, activity, regulation, and function, we can begin to understand the significance of the diversification of myosin proteins. While myosin I and II proteins retain conserved elements of structure that may dictate their similar mechanisms of motility and actin-activated MgATPase activity, their unique structures may provide the basis for the distinct localization and regulation of the two myosin types. How does the tropomyosin-inhibited actin-binding site of myosin I differ from that of the tropomyosin-activated myosin II actin-binding site? What elements of the sites of interaction of the 110K-protein and calmodulin contribute to the conserved, light-chain dependent coupling of MgATPase activity to translocation and which confer the novel calcium regulation of dissociation in vitro? It seems that the evolutionary demand for diversification of cellular motility functions has been met, at least in the actin-based system, by the evolution of isoforms tailored in structure, activity, regulation, and localization to serve complementary needs.     

Calcium and cargoes as regulators of myosin 5a activity

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.     

Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.     

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.     

Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

The influence of trace amount of calponin on the smooth muscle myosins in different states

Calponin (CaP), a thin filament-associated protein, plays an important role in the regulation of smooth muscle contractility. It has been known that CaP inhibits the actin-activated myosin MgATPase activity via binding to F-actin, and stimulates myosin MgATPase activity via binding to myosin. Our recent study revealed a new phenomenon that trace amount of CaP (TAC) could influence the function of different states of myosin. Our data showed that in the absence of actin, CaP, even in the concentration of 0.0001 microM, significantly increased the precipitations of 1 microM unphosphorylated myosin, Ca(2+)-CaM dependently, and independently phosphorylated myosin by MLCK, and stimulated the MgATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relative selective effect of TAC. In the presence of actin, myosin, and TAC, the increase of myosin precipitation was abolished, and no obvious changes of actin precipitations and actin-activated myosin MgATPase activities were observed implicating the highly efficiency of TAC on myosin being present in the absence of actin. Although we cannot give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present in the regulation of the function of myosin when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction between TAC and myosin can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.     

Heat resistance of MgATPase and contractility in muscle models

1. 1.|During the heating of a synthetic actomyosin suspension, the following sequence of events were observed. First, the rate of superprecipitation decreased; secondly the extent of superprecipitation decreased and finally the MgATPase activity was inhibited. At the same time the dissociating capability of actomyosin decreased in a solution of high ionic strength.

2. 2.|A similar lack of coincidence between the mechanical and the enzymatic activities of actomyosin was observed with an increasing proportion of inactivated myosin occurring in the reconstructed actomyosin complex.

3. 3.|The different heat resistance of contractility and MgATPase activity in muscle models may be caused by inactivated myosin bridges which form in the course of heat treatment so that the dissociating capacity of actomyosin in the presence of ATP is lost.

Unphosphorylated calf thymus and aorta myosins contract ghost myofibrils

The MgATPase activity of unphosphorylated gizzard myosin is not stimulated by actin, but the MgATPase activities of unphosphorylated calf thymus and calf aorta myosins are stimulated by actin. This suggested that unphosphorylated thymus and aorta myosins, but not unphosphorylated gizzard myosin, should be able to cause movement. The contractile activities of these myosins were examined using "ghost" myofibrils, skeletal muscle myofibrils which have been depleted of myosin. Ghost myofibrils were reconstituted with unphosphorylated and phosphorylated turkey gizzard, calf aorta, and calf thymus myosins. While ghost myofibrils reconstituted with unphosphorylated gizzard myosin did not contract, those reconstituted with unphosphorylated thymus and aorta myosins did contract. All three phosphorylated myosins supported contraction.     

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.     

Reaction of Retinol with Peroxynitrite

The reactivity of retinol with peroxynitrite, which is a strong oxidant and has been reported to induce several biological damages, was investigated. 13-cis-14-nitroretinol (1), 13-trans-14-nitroretinol (2), 13-apo-β-carotenone (3), retinal (4), 11,14-epoxyretinol (5), and 11,15-epoxyretinol (6) were identified as reaction products of retinol with peroxynitrite. From these results, it was observed that retinol can undergo a nitration reaction with peroxynitrite. Furthermore, the formation mechanisms of 1, 2, and 3 from retinol with peroxynitrite are discussed.     

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle modified by peroxynitrite

Abstract

Objective: Effect of peroxynitrite on SERCA1 activity was studied in correlation with enzyme carbonylation. Kinetic parameters and location of peroxynitrite effect on SERCA1 were determined.

Methods: Carbonyls were determined by immunoblotting. FITC, NCD-4 and Trp fluorescence were used to indicate changes in cytosolic and transmembrane regions of SERCA1.

Results: Peroxynitrite-concentration-dependent decrease of SERCA1 activity was associated with elevation of protein carbonyls. 4-HNE was not involved in carbonylation of SERCA1. Increased FITC fluorescence in the presence of peroxynitrite correlated with the decrease of the enzyme affinity to ATP.

Discussion and conclusion: Peroxynitrite-induced SERCA1 carbonylation that was not accompanied with the formation of 4-HNE-SERCA1 adducts is indicative of direct oxidation of SERCA1. As assessed by FITC fluorescence and decreased affinity of the enzyme to ATP, peroxynitrite impairment was found to occur in the cytosolic ATP-binding region of SERCA1.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.     

The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase

The microvillus 110-kD protein-calmodulin complex (designated 110K-CM) shares several properties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, 110K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K+ and EDTA (0.24 mumol P1/mg per min) or in the presence of Ca++ (0.40 mumol P1/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 mumol P1/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of 110K-CM is also enhanced by the addition of 5-10 microM Ca++, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca++. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of 110K-CM's enzymatic activity in the presence of 5-10 microM Ca++ and actin. 110K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified 110K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the 110K-CM complex is functionally analogous to the mechanoenzyme myosin.     

Interaction between troponin and myosin enhances contractile activity of myosin in cardiac muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.