Limits...
Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

Show MeSH

Related in: MedlinePlus

ATP-binding to actin·S1 (α- or β-isoform). The ATP-binding properties of the recombinant human α- and β-S1 proteins with actin present were investigated using stopped flow measurements. a After rapidly mixing 0.5 mM ATP with 0.1 μM pyrene-actin·S1, for both cardiac S1 isoforms the pyrene fluorescence transient is best described by a two exponential fit. For the fast phase, the observed rate constant was (kobs) = 658 s−1 (amp = 21%) for α and kobs = 436 s−1 (amp = 31%) for β, whereas for the slow phase, a similar kobs was found for both isoforms, kobs = 61 s−1 (amp = 2.2%) for α and 64 s−1 (amp = 5%) for β. b For both α- and β-S1 the kobs of the fast phase showed a hyperbolic dependence on ATP concentration. At high [ATP], kobs for the fast phase (=  ) saturates at 1,667 for α (filled square) and at 1,432 s−1 for β (filled triangle) with a half maximal kobs at 769 and 1,075 μM ATP (= 1/) for α and β, respectively. The slower phase is virtually independent of ATP concentration k+α1 = 40–60 s−1 for both α (open square) and β (open triangle)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375423&req=5

Fig3: ATP-binding to actin·S1 (α- or β-isoform). The ATP-binding properties of the recombinant human α- and β-S1 proteins with actin present were investigated using stopped flow measurements. a After rapidly mixing 0.5 mM ATP with 0.1 μM pyrene-actin·S1, for both cardiac S1 isoforms the pyrene fluorescence transient is best described by a two exponential fit. For the fast phase, the observed rate constant was (kobs) = 658 s−1 (amp = 21%) for α and kobs = 436 s−1 (amp = 31%) for β, whereas for the slow phase, a similar kobs was found for both isoforms, kobs = 61 s−1 (amp = 2.2%) for α and 64 s−1 (amp = 5%) for β. b For both α- and β-S1 the kobs of the fast phase showed a hyperbolic dependence on ATP concentration. At high [ATP], kobs for the fast phase (=  ) saturates at 1,667 for α (filled square) and at 1,432 s−1 for β (filled triangle) with a half maximal kobs at 769 and 1,075 μM ATP (= 1/) for α and β, respectively. The slower phase is virtually independent of ATP concentration k+α1 = 40–60 s−1 for both α (open square) and β (open triangle)

Mentions: For Figs. 3b, 4d, and 6b, the dependence of kobs on ATP concentration is defined by:3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ k_{\text{obs}} = k_{ \max } \left[ {\text{ATP}} \right] /\left( {K_{ 0. 5} { + }\left[ {\text{ATP}} \right]} \right) $$\end{document}where kmax is the maximum value of kobs and K0.5 is the nucleotide concentration required to give kobs = kmax/2.


Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

ATP-binding to actin·S1 (α- or β-isoform). The ATP-binding properties of the recombinant human α- and β-S1 proteins with actin present were investigated using stopped flow measurements. a After rapidly mixing 0.5 mM ATP with 0.1 μM pyrene-actin·S1, for both cardiac S1 isoforms the pyrene fluorescence transient is best described by a two exponential fit. For the fast phase, the observed rate constant was (kobs) = 658 s−1 (amp = 21%) for α and kobs = 436 s−1 (amp = 31%) for β, whereas for the slow phase, a similar kobs was found for both isoforms, kobs = 61 s−1 (amp = 2.2%) for α and 64 s−1 (amp = 5%) for β. b For both α- and β-S1 the kobs of the fast phase showed a hyperbolic dependence on ATP concentration. At high [ATP], kobs for the fast phase (=  ) saturates at 1,667 for α (filled square) and at 1,432 s−1 for β (filled triangle) with a half maximal kobs at 769 and 1,075 μM ATP (= 1/) for α and β, respectively. The slower phase is virtually independent of ATP concentration k+α1 = 40–60 s−1 for both α (open square) and β (open triangle)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375423&req=5

Fig3: ATP-binding to actin·S1 (α- or β-isoform). The ATP-binding properties of the recombinant human α- and β-S1 proteins with actin present were investigated using stopped flow measurements. a After rapidly mixing 0.5 mM ATP with 0.1 μM pyrene-actin·S1, for both cardiac S1 isoforms the pyrene fluorescence transient is best described by a two exponential fit. For the fast phase, the observed rate constant was (kobs) = 658 s−1 (amp = 21%) for α and kobs = 436 s−1 (amp = 31%) for β, whereas for the slow phase, a similar kobs was found for both isoforms, kobs = 61 s−1 (amp = 2.2%) for α and 64 s−1 (amp = 5%) for β. b For both α- and β-S1 the kobs of the fast phase showed a hyperbolic dependence on ATP concentration. At high [ATP], kobs for the fast phase (=  ) saturates at 1,667 for α (filled square) and at 1,432 s−1 for β (filled triangle) with a half maximal kobs at 769 and 1,075 μM ATP (= 1/) for α and β, respectively. The slower phase is virtually independent of ATP concentration k+α1 = 40–60 s−1 for both α (open square) and β (open triangle)
Mentions: For Figs. 3b, 4d, and 6b, the dependence of kobs on ATP concentration is defined by:3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ k_{\text{obs}} = k_{ \max } \left[ {\text{ATP}} \right] /\left( {K_{ 0. 5} { + }\left[ {\text{ATP}} \right]} \right) $$\end{document}where kmax is the maximum value of kobs and K0.5 is the nucleotide concentration required to give kobs = kmax/2.

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

Show MeSH
Related in: MedlinePlus