Role of actin in the myosin-linked Ca(2+)-regulation of ATP-dependent interaction between actin and myosin of a lower eukaryote, Physarum polycephalum.

Actin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend, Pharmacol. Sci. 11, 433-435]. The activity of dephosphorylated myosin remained at a low level both in the presence and absence of Ca2+, although Ca(2+)-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum, myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo. Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin. Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955-957].(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of N-ethylmaleimide on Ca-inhibition of Physarum myosin

We have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by microM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500,000 Mr) must be modified. These residues are probably the "reactive thiols" which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

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.     

Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum.

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca2+-ATPase and Mg2+-ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave Km and V values of 17.3 muM and 1.25 mumol Pi/min per mg, and 2.4 muM and 0.12 mumol Pi/min per mg, respectively. Adenylyl imidodiphosphate, a beta-gamma-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with Ki values equal to 350 and 12 muM in the presence of Ca2+ and Mg2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate (t1/2 = 24 h) by the ATP analog, 6,6'-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate (t1/2 approximately 10 min) skeletal and cardiac myosins rapidly by reacting with key cysteines.     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin

The influence of Ca2+ on the enzymatic and physical properties of smooth muscle myosin was studied. The actin-activated ATPase activity of phosphorylated gizzard myosin and heavy meromyosin is higher in the presence of Ca2+ than in its absence, but this effect is found only at lower MgCl2 concentrations. As the MgCl2 concentration is increased, Ca2+ sensitivity is decreased. The concentration of Ca2+ necessary to activate ATPase activity is higher than that required to saturate calmodulin. The similarity of the pCa dependence of ATPase activity and of Ca2+ binding to myosin and the competition by Mg2+ indicate that these effects involved the Ca2+-Mg2+ binding sites of gizzard myosin. For the actin dependence of ATPase activity of phosphorylated myosin at low concentrations of MgCl2, both Vmax and Ka are influenced by Ca2+. The formation of small polymers by phosphorylated myosin in the presence of Ca2+ could account for the alteration in the affinity for actin. For the actin dependence of phosphorylated heavy meromyosin at low MgCl2 concentrations, Ca2+ induces only an increase in Vmax. To detect alterations in physical properties, two techniques were used: viscosity and limited papain hydrolysis. For dephosphorylated myosin, 6 S or 10 S, Ca2+-dependent effects are not detected using either technique. However, for phosphorylated myosin the decrease in viscosity corresponding to the 6 S to 10 S transition is shifted to lower KCl concentrations by the presence of Ca2+. In addition, a Ca2+ dependence of proteolysis rates is observed with phosphorylated myosin but only at low ionic strength, i.e. under conditions where myosin assumes the folded conformation.     

Ca2+ activates actin-filament sliding on scallop myosin but inhibits that on Physarum myosin

The actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by microM levels of Ca2+, the mode of which is in contrast to the activating effect of Ca2+ on scallop myosin (Kohama, K. (1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.     

Biphasic steady-state kinetics of myosin adenosine triphosphatase. Evidence for a substrate effector site

The steady-state kinetics of the K+, Ca2+, and Mg2+-activated adenosine triphosphatase (ATPase) activities of rabbit skeletal myosin were investigated in the substrate concentration range from 0.05 microM to 5 mM and found not to follow Michaelis-Menten kinetics but rather to display biphasic behavior. The Ca2+-ATPase activity of myosin chymotryptic subfragment-1 (S-1), which has only one active site, also exhibits biphasic kinetics, thus excluding the possibility that the biphasic behavior is caused by negative cooperativity between the two active sites of myosin. Myosin K+ and Mg2+-ATPase are both activated by 5'-adenyl methylenediphosphonate (AdoPP[CH2]P) in a competitive manner at high substrate concentrations; i.e. the maximal velocity observed at high substrate concentrations is independent of the AdoPP[CH2]P concentration. This result provides evidence for substrate activation via binding to a regulatory site. Pyrophosphate inhibits myosin ATPase in a competitive manner at low substrate concentrations and in an uncompetitive manner at high substrate concentrations, with the uncompetitive Ki being smaller than the competitive Ki; i.e. pyrophosphate binds more tightly to the effector site than to the active site.     

Ca2+-sensitivity of actomyosin ATPase purified from Physarum polycephalum.

A contractile protein closely resembling natural actomyosin (myosin B) of rabbit skeletal muscle was extracted from plasmodia of the slime mold, Physarum polycephalum, by protecting the SH-groups with beta-mercaptoethanol or dithiothreitol. Superprecipitation of the protein induced by Mg2+-ATP at low ionic strength was observed only in the presence of very low concentrations of free Ca2+ ions, and the Mg2+-ATPase [EC 3.6.1.3] reaction was activated 2- to 6-fold by 1 muM of free Ca2+ ions. Crude myosin and actin fractions were separated by centrifuging plasmodium myosin B in the presence of Mg2+-PPi at high ionic strength. The crude myosin showed both EDTA- and Ca2+-activated ATPase activities. The Mg2+-ATPase activity of crude myosin from plasmodia was markedly activated by the addition of pure F-actin from rabbit skeletal muscle. Addition of the F-action-regulatory protein complex prepared from rabbit skeletal muscle as well as the actin fraction of plasmodium caused the same degree of activation as the addition of pure F-actin only in the presence of very low concentrations of Ca2+ ion     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of myosin from amoebae of Physarum polycephalum

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

Stimulation of calcium pump activity in heart sarcolemma by timolol

The effects of beta-adrenergic blocking agents, timolol and atenolol (1-1000 microM), were studied on rat heart sarcolemmal ATPase and Ca2+ binding activities. Timolol, unlike atenolol, increased both Ca2+-stimulated ATPase and ATP-dependent Ca2+ binding; the maximal effects were seen at 1 microM concentration of timolol. Both timolol and atenolol did not alter the sarcolemmal Mg2+ ATPase and nonspecific Ca2+ binding activities. Sarcolemmal Ca2+-stimulated ATPase was also activated by concanavalin A (6-66 micrograms/mL) which is known to alter membrane fluidity; however, Mg2+ ATPase was unaffected by this agent. These results indicate that timolol may stimulate Ca2+ pump activity in heart sarcolemma by changing membrane fluidity in a manner similar to that of concanavalin A.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.