Native Tropomyosin Research Articles

Identification of myosin A, actin and native tropomyosin constituting of arterial contractile protein (myosin B) and their characteristics.

In order to clarify the characteristic features of arterial contractile protein, natural actomyosin (myosin B), myosin A, actin and native tropomyosin were extracted from bovine carotid arteries and their characteristic features were compared with those from bovine cardiac muscle and from rabbit's skeletal muscle. In this study, the following points were clarified: l) Arterial contractile protein mainly consisted of myosin A, actin and native tropomyosin, regulatory protein respondent to Ca2+ just as having been observed in those from striated muscle. 2) Arterial myosin A, actin and native tropomyosin possessed the similar features characteristic of myosin A, act-in and native tropomyosin from striated muscle, respectively, as shown with following items; effect of divalent cations on ATP-ase activity of myosin A, GnF transformation in actin the formation of arrow-head m F-actin by reaction with arterial and skeletal H-meromyosins and the demonstration of the intraction of myosin A and F-actin with regardless of their origins. 3) The optimal concentration of Mg2+ necessary for the interaction of myosin A and F-actin was much high (10-20 mM) in myosin A from arterial wall and very low (< 0.5 mM) in myosin A from striated muscle regardless of origin of the F-actin. 4) In electronmicrographic structures, arterial myosin aggregates were shorter in the length compared with that of striated muscle, having rod shape without tapering and possessed the fine projections only at the both ends. However, any difference has not been observed between arterial F-actin and F-actin from striated muscle which held helical structure with the pitch of about 350 A and the width of about 80 A. 5) The thin filaments containing actin were located not only at muscular layer of arterial wall but also at endothelium of renal capillary. Through the above findings, it was understood that the contraction - relaxation of arterial wall, even in capillary, contributory to the performance of blood circulation was based on the association - dissociation of myosin aggregate and F-actin together with native tropomyosin respondent to Ca2+ through increase - decrease of Ca2+, which is associated with hydrolysis of ATP in the presence of Mg2+.

Interaction of SH 1 -blocked HMM with actin and ATP.

NUMEROUS studies have been performed on the effect of blocking the SH groups of myosin with sulphydryl reagents1–8. In particular, the effect of NEM has become quite clear; on blocking one specific SH group (referred to as SH1) per myosin subunit, the EDTA-ATPase of the myosin is completely inhibited whereas the Ca-ATPase is markedly activated9. However, the interaction of SH1-blocked myosin and actin has not been as thoroughly investigated. Sekine and Yanoguchi have reported that blocking SH1 with NEM does not affect the ATPase activity and superprecipitation of actomyosin10. On the other hand, the effect of native tropomyosin on the actomyosin ATPase has been reported to be abolished by NEM under certain conditions although differences were reported depending on whether myosin or actomyosin was exposed to the NEM11.

Ca 2+-binding and Ca 2+-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca 2+. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca 2+ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10 6 M −1, and 21.58·10 −6 moles·g −1. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca 2+ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca 2+. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.

Extraction of native tropomyosin-like substances from myxomycete plasmodium and the cross reaction between plasmodium F-actin and muscle native tropomyosin

1. 1.|Native tropomyosin-like substances were extracted from plasmodium of the myxomycete Physarum polycephalum, in the presence of which superprecipitation of muscle actomyosin was regulated by Ca 2+. 2. 2.|The superprecipitation of actomyosin synthesized from plasmodium F-actin and muscle myosin A was regulated by Ca 2+ in the presence of native muscle tropomyosin. 3. 3.|It was shown by sedimentation analysis and measurements of flow birefringence and viscosity that muscle tropomyosin was bound to plasmodium F-actin.

RELATIONSHIPS BETWEEN BINDING QUALITY OF MEAT AND MYOFIBRILLAR PROTEINS: IV. Contribution of Native Tropomyosin and Actin in Myosin B to Rheological Properties of Heat Set Minced-Meat Gel.

The binding quality of the heat set gel increased when F-actin was present in an appropriate ratio to myosin A. It increased further when tropomyosin was added to the myosin A/F-actin model system. The water holding capacity was increased by tropomyosin only. Thus, the contribution of not only myosin A but also of native tropomyosin and actin should be controlled in sausage manufacture. The presence of 0.6 M NaCl or KCl in the sol was necessary to produce high binding quality and high water holding capacity of heated myosin B gels. NaCl is preferred because of the taste effect on the meat product. Heating temperature (65, 80, or 90°CC) affected the physical properties of heated myosin B gels. The water holding capacity and the maximum relaxation time decreased with increasing temperature. In the manufacture of meat products, the heating temperature should be selected in accordance with the desired properties in the finished product.

The Ca 2 + sensitivity of actomyosin in solution

Actomyosin dissolved in 0.6 M KCl showed a response to Ca 2+ in the presence of MgATP and native tropomyosin (troponin-tropomyosin complex); the Mg 2+-ATPase activity was inhibited by some 20% by ethyleneglycol-bis(aminoethylether)- N, N′-tetraacetic acid (EGTA), the recovery of the viscosity decreased by ATP was delayed by EGTA and the same tendency was observed in dynamic rigidity measurements. These facts suggest that there exist some interactions between the myosin monomer and F-actin filaments in the presence of ATP and 0.6 M KCl which are regulated by Ca 2+ under the influence of the troponin-tropomyosin complex.

Participation of native tropomyosin in the ATP-contraction of an intestinal glycerinated muscle bundle.

Observations were made on the Ca-sensitivity of glycerinated muscle bundles from dog intestine. The results obtained were as follows:1) The Ca-sensitivity of glycerinated muscle bundles depends on theduration of glycerol extraction. With long-term extraction, the muscle bundles lost a large part of the Ca-sensitivity of the ATP contractility.2) When the bundles that had a typical Ca-sensitivity were treated with a low concentration of trypsin, their ATP-contractility became insensitive to varying concentrations of Ca ions.3) On treatment with intestinal native tropomyosin, the Ca-sensitivity of the ATP-contractility of 2-year-glycerinated muscle bundles was restored, to a certain extent. With the same treatment, the Ca-sensitivity of trypsin-treated 2-year-glycerinated muscle bundle was restored to the same level as that of 2-year-glycerinated bundles.From these results, it is concluded that the Ca-sensitivity of the ATP-contractility of intestinal glycerinated muscle is due to the native tropomyosin it contains.

Relationships between Binding Quality of Meat and Myofibrillar Proteins

The viscosity change of myosin A concentrated solution with or without other com-ponents was measured as the incubation time elapsed at 30°C. The viscosity of myosin A solution increased, but that of F-actin solution did not. The shear stress at 0.04 sec-1 was not increased to 1.0 dyne/cm2 in the former, but in the latter was below 0.5 dyne/cm2. The viscosity of myosin B solution increased slightly, but that of native tropomyosin-free myosin B solution decreased remarkably. In both the shear stress at 0.04 sec-1 was greater than or equal to 15 dynes/cm2. The speed of the viscosity increase in the presence of 3mM pyrophosphate and 3mM MgCl2 was higher in concentrated solution of myosin B than in that of native tropomysin-free myosin B. The shear stress at 0.04 sec-1 after 6 hr at 30°C was 11.5 and 8.2 dynes/cm2, respectively. The effect of native tropomyosin and actin on the viscosity change was discussed.

Effect of skeletal muscle native tropomyosin on the interaction of amoeba actin with heavy meromyosin.

ACTIN and myosin, recently identified in protozoa and myxomycetes, are presumably responsible for the motility of these organisms1–5. Control of this motility is, however, not at all understood. In skeletal muscle, contraction is partly under the control of the protein complex, native tropomyosin which is believed to bind to the actin filaments6,7. In the presence of Ca2+, native tropomyosin allows the interaction of actin and myosin filaments to occur wiiereas in the absence of Ca2+ native tropomyosin inhibits this interaction6, probably by inhibiting the binding of actin to myosin8. No protein like native tropomyosin has yet been identified in a primitive contractile system, but we know that Acanthamoeba actin activates the ATPase of skeletal muscle heavy meromyosin (HMM) and we have used this fact to investigate the effect of skeletal muscle native tropomyosin on the interaction of Acanthamoeba actin with HMM. We have found that native tropomyosin in the presence of Ca2+ increases the activation of HMM ATPase by Acanthamoeba actin, while in the absence of Ca2+ the native tropomyosin inhibits this activation.

Native tropomyosin: Effect on the interaction of actin with heavy meromyosin and subfragment-1

In the absence of Ca ++, native tropomyosin markedly inhibits the acto-subfragment-1 ATPase as well as the acto-heavy meromyosin ATPase despite the fact that subfragment-1, in contrast to heavy meromyosin, has only a single site for ATP. Double reciprocal plots of acto-heavy meromyosin ATPase vs. actin concentration both in the presence and absence of native tropomyosin, show that the native tropomyosin has little effect on the acto-HMM-ATPase but acts mainly by affecting the binding of actin to the HMM-ATP complex.

Relation between the inhibitory action of native tropomyosin and the dual effect of magnesium on the ATPase activity of actomyosin

The effects of various concentrations of Mg 2+ on the ATPase activities of native tropomyosin-free synthetic actomyosin, myosin B, and synthetic actomyosin to which native tropomyosin was added, and on the superprecipitation of myosin B, were investigated in the presence of 6.2 × 10 −8 m Ca 2+ and 1.7 × 10 −5 m Ca 2+. At 6.2 × 10 −8 m Ca 2+, ATPase activity of synthetic actomyosin was enhanced by 5 × 10 −6 to 1 × 10 −4 m Mg 2+. Even with supramaximal amounts of Mg 2+, the ATPase activity was not inhibited below the original level attained without added Mg 2+. At 1.7 × 10 −5 m Ca 2+, the ATPase was markedly activated by Mg 2+ in concentrations lower than 10 −5 m. Further addition of Mg 2+ decreased the ATPase activity toward, but not below, the original level. Unlike synthetic actomyosin, Mg 2+-activated ATPase activity of myosin B and of synthetic actomyosin to which native tropomyosin was added falls below the original level at high Mg 2+ concentrations if Ca 2+ level is very low. This inhibitory effect of Mg 2+ is abolished by high concentrations of Ca 2+. On the basis of these and related findings, we suggest mechanisms for the inhibitory action of native tropomyosin and for the dual effect of Mg 2+.

Low-temperature extraction of a troponin-tropomyosin factor from the I-Z-I brush of striated muscle

A new form of native tropomyosin has been obtained by a short-term, low-temperature, mild extraction of a preparation of I-Z-I brushes. This protein which we have termed the troponin-tropomyosin factor (TF), appears as a tight complex of troponin and tropomyosin in that the entire complex precipitates under the isoelectric conditions which separate these two proteins in regular native tropomyosin. Differences between native tropomyosin and TF can also be detected by gel filtration and ammonium sulfate fractionation. This TF protein complex is highly active, delaying (in the absence of calcium) the onset of superprecipitation in certain actomyosin preparations for extremely long periods of time and, under standard conditions, binding large quantities of calcium. We have considered whether it might represent a more natural form of this structural and regulatory entity.

A new form of native tropomyosin.

A new form of native tropomyosin.

Natural F-actin&lt;subtitle&gt;V. Interaction with α-Actinin, Tropomyosin and Native Tropomyosin&lt;/subtitle&gt;

Natural F-actin&lt;subtitle&gt;V. Interaction with α-Actinin, Tropomyosin and Native Tropomyosin&lt;/subtitle&gt;

The influence of native tropomyosin on the ATP threshold for turbidity development of actomyosin and myofibril suspensions

By using an ATP regenerating system to maintain constant ATP concentration, a marked drop in the ATP threshold for turbidity development is observed in the presence of EGTA 2 2 Abbreviation used: EGTA, ethylene glycol bis (β-aminoethyl ether)- N,N′-tetraacetic acid. ATPase, EC 3.6.1.3; ATP phosphohydrolase. . This effect is abolished when native tropomyosin is destroyed by trypsin digestion or removed by mild alkaline washing of actomyosin. When fresh native tropomyosin is added to such modified actomyosin, it is incapable of restoring more than a small part of the turbidity threshold shift, although it restores the ATPase inhibition almost completely.

Troponin

1. A method for isolation of troponin from native tropomyosin was described. 2. Troponin in combination with tropomyosin restored the whole activity of native tropomyosin in sensitizing the interaction of myosin and actin to Ca ion. 3. Troponin was found to bind nearly 4 moles of Ca per 10 5 g, of which most were exchangeable. The result of the experiment to determine the binding constant of these Ca binding sites was explained by assuming that half of the binding sites possessed a binding constant of 1.3×10 6 M -1 and the remaining half 5×10 4 M -1 . 4. The amount of exchangeable Ca in the contractile system was mainly accounted for by the Ca-binding capacity of troponin, which was not influenced by other contractile proteins or ATP. 5. Cardiac troponin showed a much higher affinity for Sr ion than skeletal troponin. The ratio of the former affinity to the latter was in good agreement with the ratio of the sensitivity to Sr ion of a reconstituted contractile system containing cardiac troponin to that containing skeletal troponin. Based on these findings and the results described above, it was concluded that the sensitivity of a contractile system to Ca ion is solely dependent upon the affinity for Ca ion of the troponin molecule present. 6. The mechanism of troponin regulation of the interaction of actin and myosin was discussed.

The role of native tropomyosin in the calcium sensitivity of crayfish actomyosin

1. 1. Natural actomyosin extracted from crayfish tail muscle was shown to be ensitive to calcium ions in the presence of Mg ATP at low ionic strenght. The ATPase activity was enhanced by 10 −6 M Ca 2+ by 60 per cent or more at various KCl concentrations. 2. 2. Desensitized actomyosin became sensitive to calcium ions, when native tropomyosin prepared from rabbit or crayfish muscle was added. Crayfish native tropomyosin appeared to be more effective to crayfish actomyosin than to rabbit actomyosin.

Crystals of tropomyosin and native tropomyosin

Crystals of tropomyosin and native tropomyosin

Depression of myosin B by catecholamine analogs: mechanism and in vivo significance.

ARTICLESDepression of myosin B by catecholamine analogs: mechanism and in vivo significanceCR HonigCR HonigPublished Online:01 Feb 1968https://doi.org/10.1152/ajplegacy.1968.214.2.357MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited Byβ-adrenergic blockade attenuates cardiac dysfunction and myofibrillar remodelling in congestive heart failure15 January 2010 | Journal of Cellular and Molecular Medicine, Vol. 15, No. 3Impaired Intracellular Energetic Communication in Muscles from Creatine Kinase and Adenylate Kinase (M-CK/AK1) Double Knock-out MiceJournal of Biological Chemistry, Vol. 278, No. 33Altered energy coupling in rat heart mitochondria following in vivo treatment with propranololBiochemical Pharmacology, Vol. 42, No. 3Inhibition of the mitochondrial Mg2+-ATPase by propranololBiochemical Pharmacology, Vol. 34, No. 7Phosphorylation of guinea pig cardiac natural actomyosin and its effect on ATPase activityBiochemical and Biophysical Research Communications, Vol. 73, No. 3Heart contractile proteinsBiochimie, Vol. 57, No. 11-12A possible mechanism of anesthetic-induced myocardial depressionBiochemical and Biophysical Research Communications, Vol. 57, No. 1Ca2+-binding and Ca2+-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosinBiochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 275, No. 3Catecholamines.Heart, Vol. 34, No. 9Stimulability, adenosine triphosphatases and their control by cellular redox processesDie Naturwissenschaften, Vol. 58, No. 9The purification of cardiac myofibrils with Triton X-100Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 245, No. 1Drug-Macromolecular Interactions: Implications for Pharmacological ActivityABDOMINAL AORTIC ANEURYSMSThe Lancet, Vol. 293, No. 7607CATECHOLAMINES AND THE HEARTThe Lancet, Vol. 293, No. 7607Metabolism of the heart in health and disease. Part IIIAmerican Heart Journal, Vol. 77, No. 3Effect of Adrenergic Blocking Agents on the Calcium Pump of the Fragmented Cardiac Sarcoplasmic ReticulumNature, Vol. 220, No. 5162 More from this issue > Volume 214Issue 2February 1968Pages 357-364 Copyright & PermissionsCopyright © 1968 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1968.214.2.357PubMed4383977History Published online 1 February 1968 Published in print 1 February 1968 Metrics

Molecular Weight and Subunit Structure of Tropomyosin B

Abstract Sedimentation equilibrium studies on rabbit tropomyosin have indicated heterogeneity under conditions in which it has previously been considered to exist as a single molecular species. At neutral pH, solutions of tropomyosin consist of a monomer in equilibrium with higher molecular weight species. Molecular weight determinations at high ionic strength by low speed equilibrium sedimentation and osmotic pressure indicate dissociation on dilution to give a value close to 68,000 for the molecular weight at zero concentration. Estimates of the size of the smallest macromolecular species present at both low and high ionic strength were made by the high speed equilibrium technique, and these were in accord with a molecular weight of 68,000 for the native tropomyosin monomer. Determination of molecular weights in 8 m urea by equilibrium sedimentation and osmotic pressure indicate further dissociation to two subunits of molecular weight 33,500. The subunits appear to be very similar in mass and charge, and the results are consistent with a two-chain structure for the native monomer. On the hypothesis that tropomyosin consists of two adjacent α helices, the length of the molecule is approximately 437 A. Although the existence of material other than tropomyosin can be detected in both aqueous buffer and 8 m urea, the results of gel filtration, chromatography on diethylaminoethyl cellulose, and gel electrophoresis suggest that the amounts are insufficient to affect the conclusions of this study.