Circular dichroism and fluorescence studies on troponin—tropomyosin interactions

Mani, Rajam S.; McCubbin, William D.; Kay, Cyril M.

doi:10.1016/0014-5793(75)80653-3

Cited by 17 publications

(6 citation statements)

References 23 publications

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“…Protein concentrations were estimated by absorption at 280 nm using values for the absorption coefficient (A:$) and molecular weights as follows: S-1, 7.5 cm-' and 115000; heavy meromyosin, 6.5 cm-' and 350000 [16]; actin, 1l.Ocm-' [19] and 42000 [20]; tropomyosin-troponin, 6.0 cm-' (calculated from known absorption coefficients of tropomyosin and troponin [21]; for regulated actin, 8.75 cm-' and 63 000 (calculated from the known absorption coefficients, and the molecular weight based on known values and a molar ratio of 1 tropomyosin-troponin/ 7 actin monomers).…”

Section: Methodsmentioning

confidence: 99%

Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains

Wagner¹,

Slater²,

Pope³

et al. 1979

European Journal of Biochemistry

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The actin-activated ATPase activity of myosin subfragment 1 (S-1) isoenzymes, separated on the basis of their alkali light chain (A1 and A2) content, has been analysed under a number of different conditions. Previous experiments on the effects of increasing actin concentrations on the ATPase have shown that the maximum turnover rate of ATP by S-1 (A2) was about twice that of S-1 (Al), and the K , for actin was also considerably larger for this isoenzyme. Here we show that these kinetic differences are maintained when regulated actin (actin + tropomyosin + troponin) is used in place of purified F-actin. However, while increasing the ionic strength of the solution from 6 mM to 46 mM KCl has little effect on V for S-1 (A2), the corresponding value for S-1 (Al) increases to approximately equal that of S-1 (A2), suggesting that the two isoenzymes are controlled by the same rate-limiting process in the steady state under these conditions. The value of K , also increases with increasing ionic strength for the two isoenzymes, but that for S-1 (Al) increases more rapidly and approaches that for S-1 (A2). These experiments were carried out at at fixed S-1 concentration, but the maximum rate of actin activation of the S-1 ATPase can also be obtained from experiments where the actin concentration is fixed and the S-1 concentration varied. Under these conditions in 6 mM KCl, the maximum rate of ATP hydrolysis expressed per mole of actin at infinite S-1 concentration is the same for both s-1 (Al) and s-1 (A2), and this value numerically equals the maximum rate of hydrolysis of ATP by S-1 (A2) expressed at infinite actin concentration. These results suggest that the rate-limiting step in the ATPase cycle for S-1 (A2) is the release of products from an actin . S-1 . ADP . Pi complex, while for S-1 (Al), where the value of V at infinite actin concentration is considerably lower, a different process is rate limiting, and this process may occur before actin reassociation with the S-1 . ADP . Pi complex, using the Lymn-Taylor kinetic model. Thus increasing the ionic strength appears to change the rate-limiting process in the steady-state hydrolysis of ATP by actoS-1 (Al). These experiments are discussed in terms of the role of the alkali light chains in the myosin ATPase and muscle contraction.

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Section: Methodsmentioning

confidence: 99%

Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains

Wagner¹,

Slater²,

Pope³

et al. 1979

European Journal of Biochemistry

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“…Human Tm shows two melting transitions ( T m s), one at about 40 °C and the other at about 43 °C during the thermal‐induced unfolding monitored by CD. Previous investigations using CD of the thermal‐induced unfolding of skeletal Tm from other organisms also identified two melting transitions: rabbit Tm has T m s at 43 and 51 °C [35], and rat Tm has T m s at 30 and 44 °C [38]. Chicken smooth Tm has T m s at 32 and 44 °C as determined by DSC [39].…”

Section: The Stability Of Human Tmmentioning

confidence: 99%

“…CD and DSC experiments were used as methods for evaluating the effect of mutations on the stability of Tms [34]. The thermal‐induced unfolding of the rabbit [35–37], rat, and chicken [38,39] skeletal Tms have been characterized as a multistep process with at least two melting transitions. Human Tm shows two melting transitions ( T m s), one at about 40 °C and the other at about 43 °C during the thermal‐induced unfolding monitored by CD.…”

Section: The Stability Of Human Tmmentioning

confidence: 99%

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human α‐tropomyosin

Hilario

Silva

Ramos

et al. 2004

European Journal of Biochemistry

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Mutations in the protein a-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca 2+ . However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg 2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.Keywords: circular dichroism; differential scanning calorimetry; Pichia pastoris; tropomyosin.Tropomyosins (Tms) are a family of highly conserved proteins found in most eukaryotic cells. The striated muscle isoform is an a-helical protein, which forms a parallel coiled-coil dimer twisted around the long axis of the actin filament. Each polypeptide chain has 284 amino acid residues, and each dimer binds to seven actin monomers and one troponin (Tn) complex (TnC, TnI and TnT). In striated muscle cells the Tm polymerizes in a head-to-tail fashion, and together with the troponin complex, regulates the Ca 2+ sensitivity of the actomyosin Mg 2+ ATPase complex [1]. The Tm amino acid sequence shows a sevenresidue pattern (a to g) repeated throughout the entire sequence. Positions a and d, on the same side of the helices, are usually occupied by apolar amino acids that allow hydrophobic interactions between chains. Positions e and g are often occupied by charged residues, and therefore contribute to the stabilization of the parallel coiled-coil structure by ionic interactions with residues at positions e¢ and g¢ of the other helix. Positions b, c and f are occupied by polar or ionic residues and they interact with solvent or other proteins [1]. In addition to the heptapeptide repeat, there are seven consecutive repetitions of approximately 40 residues each in the entire length of the chain, which correspond to the actin binding sites [2].Recom...

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“…Protein concentrations were determined spectrophotometrically on a Cary 118C instrument employing previously established extinction coefficients for the cardiac [12] and skeletal [13] troponins and tropomyosins.…”

Section: Protein Concentrationsmentioning

confidence: 99%

A circular dichroism and biological activity study on the hybrid species formed from bovine cardiac and rabbit skeletal troponin subunits

1977

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Circular dichroism and fluorescence studies on troponin—tropomyosin interactions

Cited by 17 publications

References 23 publications

Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains

Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human α‐tropomyosin

A circular dichroism and biological activity study on the hybrid species formed from bovine cardiac and rabbit skeletal troponin subunits

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