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)     

Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

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.     

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.     

Effects of phosphorylation, magnesium, and filament assembly on actin-activated ATPase of pig urinary bladder myosin

The relationship between the light-chain phosphorylation and the actin-activated ATPase activity of pig urinary bladder myosin was either linear or nonlinear depending on the free Mg2+ concentration. Varying the free [Mg2+] in the presence of 50 mM ionic strength (I) had a biphasic effect on the actin-activated ATPase. In 100 mM I, the activity increased on raising the free [Mg2+]. The activity of the phosphorylated myosin was 3-23-fold higher than that of the unphosphorylated myosin at all concentrations of free Mg2+, pH, and temperature used in this study. The increase in the turbidity and sedimentability of both phosphorylated and unphosphorylated myosins on raising the free [Mg2+] was associated with a rise in the actin-activated ATPase activity. However, myosin light-chain phosphorylation still had a remarkable effect on the actin activation. The myosin polymers formed under these conditions were sedimented by centrifugation. Experiments performed with myosin polymers formed in mixtures of unphosphorylated and phosphorylated myosins showed that the presence of phosphorylated myosin in these mixtures had a slight effect on the sedimentation of the unphosphorylated myosin but it had no effect on the actin-activated ATP hydrolysis. Electron microscopy showed that the unphosphorylated myosin formed unorganized aggregates while phosphorylated myosin molecules assembled into bipolar filaments with tapered ends. These data show that although the unphosphorylated and phosphorylated myosins have the same level of sedimentability and turbidity, the filament assembly present only with the phosphorylated myosin can be associated with the maximal actin activation of Mg-ATPase.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Effects of removal of light chain 2 on the ATPase activities of cardiac myosin from normal and thyrotoxic rabbits

Steady-state ATPase activities of cardiac myosin from thyroxine-treated rabbit hearts have been determined before and after removal of the 18-kDa light-chain subunit (LC2) of myosin. LC2 was selectively removed from myosin by treatment with a myofibrillar protease according to the method of Kuo and Bhan (Biochem. Biophys. Res. Commun. 92, 570-576 (1980) ). The effects of removal of LC2 on the enzymatic properties of thyrotoxic myosin were compared with the results obtained for cardiac myosin from normal rabbits by parallel studies. It has been found that removal of LC2 does not affect the Ca2+- and K+ (EDTA)-ATPase activities of these myosins. The actin-activated myosin Mg2+-ATPase activities of intact and LC2-deficient thyrotoxic myosin were 0.18 +/- 0.03 and 0.36 +/- 0.03 mumol Pi/mg per min, respectively, whereas the actin-activated myosin Mg2+-ATPase activities of intact and LC2-deficient normal myosin were 0.12 +/- 0.02 and 0.18 +/- 0.03 mumol Pi/mg per min, respectively. Thus, removal of LC2 increases the actin-activated myosin Mg2+-ATPase activity of thyrotoxic myosin by 100%, and the same activity is increased about 50% for normal myosin, indicating that the degree of potentiation of actin-activated myosin Mg2+-ATPase activity as a result of LC2 removal is 2-fold greater in thyrotoxic myosin than that obtained for normal myosin. These results suggest that LC2 does not influence the increased actomyosin ATPase activity of thyrotoxic myosin and that potentiation of actomyosin ATPase following LC2 removal may depend on the variations of the heavy-chain domain where LC2 interacts.     

Enzymatic comparisons between light chain isozymes of human cardiac myosin subfragment-1

Human cardiac ventricular myosin subfragment-1 (S-1) was prepared by chymotryptic digestion of myosin purified from adult and fetal hearts. The enzymatic properties of adult S-1 were compared to those of two light chain isozymes of fetal S-1 which were separated by ion-exchange chromatography. One fetal isozyme contained a light chain (LC) indistinguishable from the adult ventricular LC1 and the other fetal isozyme contained the LC1 variant that is a component of intact fetal myosin. The fetal isozymes had identical actin-activated Mg2+ ATPase rates at all actin concentrations, as well as the same K+EDTA, Ca2+, and Mg2+ATPase rates. Furthermore, both fetal isozymes had the same actin-activated Mg2+ATPase rates as S-1 purified from adult hearts. The K+EDTA and Ca2+ATPase rates of adult S-1 were only slightly different from those of fetal S-1. These observations are consistent with other available data suggesting that human fetal and adult ventricular myosin differ only in light chain content, not in heavy chain composition, and indicate that isozymic LC1 variation does not alter the steady-state ATPase rate of human cardiac S-1.     

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.     

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)     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

Functional integrity of the SH1 site in myosin from hypertrophied myocardium.

The hypothesis that an alteration in the SH1 site of hypertrophy myosin is reponsible for the reduced Ca2+-stimulated ATPase activity is examined.The functional integrity of the SH1 site was evaluated by measurement of the (K+)-EDTA-stimulated and Mg2+-inhibited ATPase activities. Neither activity differed from control although the Ca2+-stimulated ATPase of the same preparations was significantly reduced. The reduction in Ca2+-activated ATPase was independent of ionic strength. Titration with N-ethylmaleimide elevated the Ca2+-stimulated ATPase of hypertrophy myosin to the same peak activity as control. Actin-stimulated ATPase activity of hypertrophy myosin was also reduced. The results indicate that the SH1 of hypertrophy myosin is functionally intact for (K+)EDTA-stimulated ATPase and Mg2+ inhibition, but functionally deficient with regard to Ca2+-stimulated and actin-activated ATPase activities. This implies a partition of the functional aspects of SH1.     

Phosphorylation of myosin light chain and the actin-activated ATPase activity of adrenal medullary myosin

Myosin light chain kinase was partially purified from bovine adrenal medulla. A polypeptide of Mr 165,000 dalton was identified as kinase by using anti-gizzard myosin light chain kinase IgG on immunoreplica. Phosphorylation of medullary myosin was Ca2+- and calmodulin-dependent. The phosphorylated myosin was showed to enhance the actin-activated Mg2+-ATPase activity. In contrast, the myosin ATPase activity was dramatically decreased by dephosphorylation of myosin.     

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.     

Studies on the amino groups of myosin ATPase. Trinitrophenylation of reactive lysyl residue in ventricular and atrial myosins

Myosin and heavy meromyosin from ventricular, atrial, and skeletal muscle were purified and trinitrophenylated by 2,4,6-trinitrobenzene sulfonate. The trinitrophenylation reaction followed a complex kinetics consisting of a fast and slow reaction in all preparations studied. Reactive lysine residues were trinitrophenylated during the fast reaction with a concomitant decrease in K⁺ (EDTA)-activated ATPase and an increase in Mg²⁺-stimulated ATPase activities of myosin. The extent of increase in Mg²⁺-mediated ATPase was the highest with skeletal and the lowest with atrial myosin. The trinitrophenylation of the less reactive lysyl residues continued during the slow reaction. The rate constants of the reactions and the number of reactive lysine residues were evaluated by computer analyses of the trinitrophenylation curves. Two reactive lysine residues were found in skeletal and ventricular myosins while their number in atrial myosin was somewhat lower. The rate of trinitrophenylation in skeletal muscle myosin or heavy meromyosin was always higher than in the two cardiac myosin isozymes. Addition of KCl increased the trinitrophenylation of both highly reactive and slowly reactive lysyl residues in all of the three heavy meromyosins, however, the effect was more profound with cardiac heavy meromyosins. Addition of MgADP induced spectral changes in trinitrophenylated skeletal but not in cardiac myosins. Similar changes occurred in skeletal and to a lesser degree in ventricular heavy meromyosin, but no definite spectral changes were observed in atrial heavy meromyosin. The findings suggest that structural differences exist around the reactive lysyl residue in the head portion of the three myosins.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.     

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.     

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.     

A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.     

Identical behavior of the two active sites of myosin with respect to trinitrophenylation.

Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.