A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized-actomyosin

1. The preparation and properties of a myofibrillar protein factor which inhibits the Mg(2+)-activated adenosine triphosphatase of desensitized actomyosin is described. 2. This factor had negligible effect on the Mg(2+)-activated adenosine triphosphatase of natural actomyosin and on the Ca(2+)-activated adenosine triphosphatases of desensitized actomyosin and myosin. 3. The Mg(2+)-activated inosine triphosphatase activity of desensitized actomyosin was not affected by the factor. 4. The inhibitory effect was sensitive to ionic strength. In addition to their ionic effects Mg(2+) and Ca(2+) appeared to have a specific action in reducing the effect of the inhibitor. 5. F-actin reduced the inhibition whereas Bailey-type tropo-myosin had little effect. 6. As far as can be judged from the reported experiments this factor is different from any of the previously described myofibrillar components.     

The adenosine-triphosphatase activity of dissociated acto-heavy-meromyosin

1. At low ionic strength, when turbidity and viscosity measurements indicated dissociation of acto-heavy-meromyosin, its adenosine triphosphatase was strongly activated by Mg(2+) and Ca(2+). 2. The characteristics of the adenosine triphosphatase of dissociated acto-heavy-meromyosin in the presence of Mg(2+) were similar to those reported for myofibrils and actomyosin. 3. In the presence of Ca(2+) the adenosine-triphosphatase activity was much less sensitive to ionic strength than was the case with Mg(2+). 4. At low ionic strength Mg(2+) was more effective in maintaining the dissociation of acto-heavy-meromyosin in the presence of ATP than was Ca(2+). This difference was not apparent when ATP was replaced by ITP. 5. Although the recovery of viscosity was complete on reassociation of acto-heavy-meromyosin the turbidity did not return to the original value. 6. The general implications of Mg(2+) activation of acto-heavy-meromyosin when classical interpretation indicates dissociation of the complex are discussed.     

The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin

1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca²⁺-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100° and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca²⁺-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca²⁺-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca²⁺-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.     

The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.     

The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca²⁺-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca²⁺ in the medium. 4. The Ca²⁺-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca²⁺-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.     

The subunits and biological activity of polymorphic forms of tropomyosin

1. Free thiol groups were shown to be essential for tropomyosin to effect maximum inhibition of the Ca(2+)-stimulated ATPase (adenosine triphosphatase) of desensitized actomyosin but not for its activity in the regulatory-protein system. 2. The activity of tropomyosin on the Mg(2+)-stimulated ATPase in the regulatory-protein system was more susceptible to enzymic digestion and thermal denaturation than its effect on the Ca(2+)-stimulated ATPase of actomyosin. 3. Rabbit skeletal tropomyosin migrated as two distinct electrophoretic components in the presence of sodium dodecyl sulphate and urea and as four components on isoelectric focusing in urea. 4. The two main subunits present in rabbit skeletal tropomyosin, which have been named the alpha- and beta-chains, were separated by chromatography on CM-cellulose in urea at pH4.0. They were shown to be virtually identical in amino acid composition, except for their cysteine contents. The alpha(2) and beta(2) forms of tropomyosin possessed all the biological activities characteristic of normal tropomyosin preparations. 5. In skeletal muscle the alpha and beta components of tropomyosin were present in the proportion of 4:1. Somewhat lower ratios were obtained in skeletal muscle of sheep, pig and cow. 6. Tropomyosin isolated from cardiac muscle and Pecten maximus adductor muscle migrated as one band only. These tropomyosins possessed similar biological activities to those isolated from skeletal muscle.     

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 enzymic properties of a modified ox heart myosin adenosine triphosphatase on covalent binding to an insoluble cellulose matrix

The preparation of ox heart myosin and its partial digestion with cellulose-bound papain is described. A procedure is outlined by which heavy meromyosin subfragment 1 can be covalently bound to a cellulose ion-exchange matrix. Attachment of heavy meromyosin subfragment 1 to the insoluble matrix results in a change in the ion specificity towards ATP hydrolysis. Unlike the soluble enzyme the bound form is activated by both Ca(2+) and Mg(2+). Maximal activation by Ca(2+) occurred at a lower concentration for the bound enzyme. Mg(2+) activates at a concentration which causes near-maximal inhibition of the Ca(2+)-activated adenosine triphosphatase (ATPase) of the non-bound enzyme. The Mg(2+)-activated ATPase of the bound enzyme was in turn inhibited by the presence of Ca(2+). The activation by Mg(2+) resembles the characteristic enzymic action of the actin-subfragment 1 complex.     

Preparation and general properties of a soluble adenosine triphosphatase from mitochondria

1. The purification of an adenosine triphosphatase present in aqueous extracts of acetone-dried ox-heart mitochondria is described. 2. No evidence was found for the presence of more than one protein having adenosine-triphosphatase activity in these extracts. 3. The enzyme is less stable at 0 degrees than at 25 degrees but is stabilized by glycerol. 4. The activity is dependent on the presence of Mg(2+) or certain other bivalent metal cations. 5. The adenosine-triphosphatase activity of the Mg(2+)-activated enzyme is enhanced by 2,4-dinitrophenol. 6. The kinetics of Mg(2+) activation indicate that the ATP-Mg(2+) complex is the important substrate: free ATP and Mg(2+) are inhibitory. 7. This preparation of mitochondrial adenosine triphosphatase has many properties in common with the adenosine triphosphatase coupling factor from mitochondria (Racker, 1961).     

The regulatory proteins of the myofibril. Characterization and properties of the inhibitory factor (troponin B)

1. Gel-filtration results indicate that the major component of inhibitory-factor preparations isolated by dissociation of the troponin complex consisted of a protein of subunit weight 23000 daltons. By the same procedure a molecular weight of 18000 was obtained for the calcium-sensitizing factor. 2. The inhibitory factor is specific for the actomyosin type of ATPase and ITPase. It is effective on desensitized actomyosin, natural actomyosin and intact myofibrils. 3. For inhibition, the actomyosin ATPase must be stimulated by Mg(2+), Ca(2+) or Mn(2+). The Co(2+)-, Cd(2+)- or Zn(2+)-stimulated ATPases are not affected. 4. Biological activity is stable to treatment with dissociating agents, heat, pH11, pH1 and carboxymethylation. 5. Increasing amounts of actin, but not myosin or tropomyosin, progressively neutralize the inhibitory activity when added to desensitized actomyosin or myofibrils.     

The biological activity of subfragment 1 prepared from heavy meromyosin

1. The action of trypsin, chymotrypsin and subtilisin on the adenosine-triphosphatase and actin-combining activities, as measured by viscometric means, of H-meromyosin were compared. 2. Subfragment 1 produced by prolonged tryptic digestion has a molecular weight of 129000. 3. The preparations isolated by gel filtration and actin combination were shown to be similar. 4. Subfragment-1 preparations possess appreciably higher adenosine-triphosphatase activities than H-meromyosin when related to total nitrogen. 5. Chromatographic and gelfiltration studies indicated that adenosine-triphosphatase activity is not distributed uniformly in all fractions of subfragment 1. 6. The Ca(2+)-activated adenosine triphosphatase of subfragment 1 was stimulated by thiol reagents in a similar fashion to myosin and H-meromyosin. 7. Subfragment 1 differed from myosin and H-meromyosin in that its adenosine triphosphatase was only slightly activated by Mg(2+) in the presence of actin. 8. A subfragment-1-like component was obtained by chymotryptic digestion of H-meromyosin. 9. The results obtained from enzymic and hydrodynamic studies and from amino acid analyses are compatible with the concept of one molecule of H-meromyosin giving rise to one molecule of subfragment 1 on proteolytic digestion.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Effect of bivalent cations on the adenosine triphosphatase of actomyosin and its modification by tropomyosin and troponin

1. After removal of tropomyosin and troponin from the `natural'' actomyosin complex, the adenosine triphosphatase activity of the resulting `desensitized'' actomyosin is stimulated to the same extent by various bivalent cations with an ionic radius in the range 0·65–0·99å when tested at optimum concentration of the metal ion in the presence of 2·5mm-ATP at low ionic strength and pH7·6. Under identical conditions the adenosine triphosphatase activity of myosin alone is stimulated to an appreciable extent only by Ca²⁺ (ionic radius 0·99å). 2. Tropomyosin narrows the range of size of the stimulatory cations by inhibiting specifically the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by Ca<sup>2+</sup> or the slightly smaller Cd<sup>2+</sup> (ionic radius 0·97å). Tropomyosin has no effect on the adenosine triphosphatase activity of `desensitized'' actomyosin when stimulated by the smaller cations, nor on the Ca²⁺-activated adenosine triphosphatase activity of myosin alone. 3. The adenosine triphosphatase activity of the `natural'' actomyosin system (containing tropomyosin and troponin) stimulated by the smallest cation, Mg²⁺ (ionic radius 0·65å), is low when the system is deprived of Ca²⁺ but high in the presence of small amounts of Ca²⁺. This sensitivity to Ca²⁺ seems to be a unique feature of the Mg²⁺-stimulated system. 4. The changes in specificity of the myosin adenosine triphosphatase activity in its requirement for bivalent cations caused by interaction with actin, tropomyosin and troponin primarily concern the size of the metal ions. The effects on enzymic properties of myofibrils due to tropomyosin and troponin can be demonstrated at low and at physiological ionic strength.     

The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit.

1. A series of defined peptides which span the complete sequence were produced from troponin I isolated from white skeletal muscle of the rabbit. 2. Two peptides, CF1 (residues 64-133) and CN4 (residues 96-117) inhibited the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition was potentiated by tropomyosin and the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition, unlike that of troponin I and peptides derived from it, was not potentiated by tropomyosin. 4. The most active inhibitor, peptide CN4, was 45-75% as effective as troponin I when compared on a molar basis. The inhibitory peptide, CN4, and also whole troponin I were shown by affinity chromatography to interact specifically with actin. 5. A strong interaction with troponin C was demonstrated with peptide CF2 (residues 1-47), from the N-terminal region of troponin I. Somewhat weaker interactions were shown with peptides CN5 (residues 1-21) and with the inhibitory peptide CN4. 6. The significance of these interactions for the mechanisms of action of troponin I is discussed.     

Evidence of a new type of protein-protein interaction: desensitized actomyosin blocks Ca(2+)-sensitivity of the natural one. A possible model for an intracellular signalling system related to actin filaments

Actin filaments are certainly believed to function as an intracellular signalling system; however, this is not confirmed by direct evidence. We used a two-layer actomyosin gel with a concentration gradient of the troponin-tropomyosin complex (TT-complex, Ca(2+)-sensitive system) between the two layers. To prepare one layer of the system, natural actomyosin (nAM) rich in TT-complex was used. To prepare the second layer, we used desensitized actomyosin (dAM) without the complex. All experimental studies were made in medium with a low ionic strength. Two phenomena were observed: (1) dAM blocks Ca(2+)-sensitivity of nAM when the dAM weight portion in the system (as well as in mixed nAM + dAM suspension) reaches 40% and more; further increase of the dAM portion does not affect the Ca(2+)-sensitivity; (2) it was electrophoretically shown that a rapid diffusion of the TT-complex from nAM gel into the dAM gel took place. The apparent diffusion coefficient for the TT-complex in dAM gel is about (1-4).10(-4) cm2/sec, i.e. three orders higher than the same values for protein diffusion in water.     

Binding of the Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli to phospholipid vesicles.

Incubation of the Ca2+,Mg2+-activated adenosine triphosphatase of Escherichia coli with phospholipid vesicles resulted in binding of the enzyme to the lipid. Binding was observed with vesicles of soybean phospholipid (asolectin), phosphatidyglycerol, phosphatidylserine, phosphatidylcholine, and cardiolpin. Binding was not affected by alterations in pH in the range of pH 6.5 to 8.5, by ionic strength, or by the presence of Mg2+. Loss of the delta subunit from the enzyme had no effect on binding. However, removal of the delta and epsilon subunits by treatment of the enzyme with trypsin prevented binding to phospholipid. This treatment also removed a small portion (less than 2000 daltons) of the alpha subunit. It is concluded that the ATPase of E. coli binds to phospholipid vesicles mainly by nonpolar interactions through the alpha and (or) epilson subunits of the enzyme.     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

Oxidative phosphorylation in Escherichia coli K 12. Mutations affecting magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.     

Dinitrophenol-stimulated adenosine triphosphatase activity in extracts of Desulfovibrio gigas

A dinitrophenol (DNP)-stimulated adenosine triphosphatase (ATPase) has been found in both the soluble and particulate fractions of the anaerobic sulfate-reducing bacterium, Desulfovibrio gigas. As the soluble ATPase was labile to storage, only the particulate enzyme was studied in detail. It was optimally stimulated by DNP at 4 mm, and activity was insensitive to inhibition by ouabain. The ATPase was stimulated by both Ca(2+) and Mg(2+), but the magnitude of the stimulation was dependent upon pH. In the presence of Ca(2+) the optimum pH was 6.5, whereas, in the presence of Mg(2+) the pH optimum was 8.0. However, under optimal conditions the activity was the same with either Mg(2+) or Ca(2+). Both adenosine triphosphate and guanosine triphosphate were hydrolyzed, but activity toward guanosine triphosphate was only one-tenth that observed with adenosine triphosphate.