Fractionation of troponin into two distinct proteins

Hartshorne, D.J.; Mueller, Helmut

doi:10.1016/0006-291x(68)90610-4

Cited by 135 publications

(44 citation statements)

References 5 publications

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“…F-actin was then prepared as previously described (Fisher, Curmi, Barden & dos Remedios, 1983;Miki, Hambly & dos Remedios, 1986). Troponin-tropomyosin complex was prepared from acetone powder (Hartshorne & Mueller, 1968). Ca2+-regulated F-actin filaments were made by reconstituting Factin with the native troponin-tropomyosin complex in the molar ratio 7: 1 as previously described (Murray, 1982) by combining purified F-actin with purified troponin-tropomyosin complex in the ratio 7:1: 1 corresponding to G-actin: troponin tropomyosin.…”

Section: Superprecipitationmentioning

confidence: 99%

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'‐dithiobis(2‐nitrobenzoic acid).

Wilson

Remedios

Stephenson

et al. 1991

The Journal of Physiology

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SUMMARY1. The sulphydryl groups of skinned skeletal muscle fibres have been reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in order to determine whether the effects of modifications to the contractile proteins are reflected in changes in the physiological properties of the contractile apparatus and Ca2+-regulatory system. 2. Results obtained from fast-twitch and slow-twitch rat fibres which were treated with DTNB (10 mm, pH 8-6, 5 C) for various periods of time under relaxing conditions showed that a major effect of the modification was to reduce the level of maximally Ca2+-activated force and fibre stiffness. Force and fibre stiffness were found to decline in proportion. Treatment with DTNB under these conditions did not cause a rise in force or fibre stiffness in relaxed fibres of either type.3. The effects induced by DTNB under relaxing conditions were substantially reversed by exposure to the reducing agent dithiothreitol (DTT) (10 mM, pH 741, 23°C). Force abolished by 30-35 s treatment with DTNB recovered after subsequent DTT treatment to 67+3 % (mean + S.E.M., n = 4) in fast-twitch fibres and to 91 + 2 % (n = 7) in slow-twitch fibres. These results were significantly different (t test, P < 0f001) indicating that the level of force recovery depended upon the fibre type.4. DTNB was found to affect not only the maximal Ca2+-activated force, but also the force-pCa (pCa = -logl0[Ca2+]) relationships of the fibres in a complex, fibretype specific way. DTT treatment partially reversed these DTNB effects. 5. The skinned fibre preparations reacted differently with DTNB under rigor conditions than under relaxing conditions, indicating that rigor modifies the reactivity of the functional sulphydryl groups to the thiol-targeted agents.6. When superprecipitation assays (an in vitro analogue of fibre contraction) were

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Section: Superprecipitationmentioning

confidence: 99%

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'‐dithiobis(2‐nitrobenzoic acid).

Wilson

Remedios

Stephenson

et al. 1991

The Journal of Physiology

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“…However, as mentioned above, this kind of activation mechanism would not seem to produce the required variation observed experimentally in Vmax. Hartshorne & Mueller (1968) and Greaser & Gergely (1971) have recently shown that troponin which had previously been considered a homogeneous protein is in fact a mixture of proteins, three of which, according to Greaser & Gergely, are necessary to provide both inhibition of actomyosin Mg-ATPase and calcium sensitivity. The X-ray work of H. E. Huxley & Brown (1967) also poses a problem.…”

Section: Ca Effect On Muscle Force and Velocity 133mentioning

confidence: 99%

The effect of calcium on the force‐velocity relation of briefly glycerinated frog muscle fibres

Julian¹

1971

The Journal of Physiology

252

144

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SUMMARY1. Twitch fibres were isolated from the semitendinosus muscles of frogs. The sarcolemma was made more permeable by a 30 min soak in a solution containing 47-3 % glycerine (v/v), 2 mM-EGTA and 10 mm phosphate buffer, pH 7. This was followed by a 30-60 min soak in a solution containing the non-ionic detergent Lubrol-WX. The fibres were then placed in a relaxing medium containing (in mM): KCl, 100; MgCl2, 1; ATP, 4; EGTA, 2; imidazole buffer, 10; pH 7 0.2. A piece of fibre about 1-2 mm long treated as described in (1) was attached to a servo apparatus. This apparatus made it possible either to hold fibre length constant giving isometric conditions, or alternatively to hold the force constant while measuring isotonic length changes. A special network made it possible to switch control from isometric to isotonic conditions so that afterloaded contractions with a shortening stop could be carried out.3. Contractions were induced at about 40 C by lowering the pCa in the relaxing solution to various levels determined by the ratio of calcium and EGTA added. Contractions were never observed above pCa 7. The steady force generated reached a maximum over the range of pCa 6-09 to 5-49. The relationship between steady force generated and pCa is S-shaped and very steep, implying that multiple interacting binding sites for calcium are involved in the force generating process.4. The relative force-velocity relation is the same at pCa 6-09 and 5*49 where the steady force is at a maximum. The data points can be well fitted by a hyperbola in which the extrapolated value for Vmx is 2-39 muscle lengths/sec. The values obtained for the Hill parameters a/PO and b are within the range of those reported for living electrically excited frog muscle.

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“…This necessitated the separation of the troponin from the complex before the acidic precipitation method for purifying troponin could be used. Skeletal-muscle troponin, at pH 1.0 and in the presence of 1.OM-KC1, can be separated into troponin C and a complex of troponin I and troponin T (Hartshorne & Mueller, 1968). Unlike skeletalmuscle troponin, all the subunits of bovine cardiac troponin were precipitated at pH 1.0 in 1.OM-KC1, indicating that the troponin I and troponin T components of bovine cardiac troponin have surface charges different from those of their counterparts in rabbit skeletal-muscle troponin.…”

Section: Resultsmentioning

confidence: 99%

Circular-dichroic studies on the conformational behaviour of troponin and tropomyosin from bovine cardiac muscle

Lin¹,

Cassim²

1978

Biochemical Journal

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Bovine cardiac troponin is similar to rabbit skeletal troponin with respect to secondary structure, amino acid composition and molecular weight of the subunits, but differs slightly with respect to biological activity and surface charges of the subunits. Previous circular-dichroic studies of the subunits and recombination of subunits have indicated significant Ca2+-induced delocalized conformational changes. Present studies of the native troponin complex are not in accord with such changes. Furthermore the formation of the troponin-tropomyosin complex in vitro results in no delocalized conformational changes, nor does it sensitize troponin to Ca2+-induced changes. It is suggested that the troponin complex cannot be dissociated into subunits without significant and irreversible conformational perturbation.

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Fractionation of troponin into two distinct proteins

Cited by 135 publications

References 5 publications

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'‐dithiobis(2‐nitrobenzoic acid).

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'‐dithiobis(2‐nitrobenzoic acid).

The effect of calcium on the force‐velocity relation of briefly glycerinated frog muscle fibres

Circular-dichroic studies on the conformational behaviour of troponin and tropomyosin from bovine cardiac muscle

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