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Limited digestion of α -actinin in the presence of F-actin

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ABSTRACT

N-terminal actin-binding domain of α-actinin is connected to central rod domain through flexible neck region that is susceptible to proteolysis. It is suggested that the neck region assumes variable orientations by actin binding. In order to examine the effect of actin binding to α-actinin, we carried out limited digestion of α-actinin by chymotrypsin in the presence and absence of F-actin. Although the cleavage process was retarded when bound to F-actin, digestion to 32 kDa-head and 55 kDa-rod domains occurred through the same intermediate products as the digestion in the absence of F-actin. N-terminal sequencing of 55 kDa-fragment showed the neck region was cleaved at 276-Leu. The cleavage site was not affected by binding to F-actin nor ionic strength of the solvent. It was also indicated that α-actinin was cleaved at 15-Tyr by chymotrypsin. Quantitation of the cleavage products by densitometry of the SDS-gels suggested the conformational change of α-actinin at domain-connecting regions by F-actin binding.

No MeSH data available.


A schematic model of α-actinin molecule. N-terminal containing actin-binding domain (ABD) is connected to rod-domain through a short flexible segment (neck). Dimers are formed by lateral interaction between rod-domains.
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f1-7_29: A schematic model of α-actinin molecule. N-terminal containing actin-binding domain (ABD) is connected to rod-domain through a short flexible segment (neck). Dimers are formed by lateral interaction between rod-domains.

Mentions: α-Actinin is a rod shaped homodimer of polypeptides of ~100 kD each that are oriented anti-parallel1,2. Based on its domain structure, α-actinin is classified as a member of spectrin superfamily. Each subunit consists of three functional domains; N-terminal containing actin binding domain (ABD) with tandem calponin homology motifs, rod domain with four spectrin-repeats, and regulatory tail domain with EF-hand like motifs (Fig. 1). The binding activity of non-muscle α-actinins was shown to be Ca2+-sensitive13,14 while that of muscle isoforms was regulated by phosphoinositides15,16.


Limited digestion of α -actinin in the presence of F-actin
A schematic model of α-actinin molecule. N-terminal containing actin-binding domain (ABD) is connected to rod-domain through a short flexible segment (neck). Dimers are formed by lateral interaction between rod-domains.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036780&req=5

f1-7_29: A schematic model of α-actinin molecule. N-terminal containing actin-binding domain (ABD) is connected to rod-domain through a short flexible segment (neck). Dimers are formed by lateral interaction between rod-domains.
Mentions: α-Actinin is a rod shaped homodimer of polypeptides of ~100 kD each that are oriented anti-parallel1,2. Based on its domain structure, α-actinin is classified as a member of spectrin superfamily. Each subunit consists of three functional domains; N-terminal containing actin binding domain (ABD) with tandem calponin homology motifs, rod domain with four spectrin-repeats, and regulatory tail domain with EF-hand like motifs (Fig. 1). The binding activity of non-muscle α-actinins was shown to be Ca2+-sensitive13,14 while that of muscle isoforms was regulated by phosphoinositides15,16.

View Article: PubMed Central - PubMed

ABSTRACT

N-terminal actin-binding domain of α-actinin is connected to central rod domain through flexible neck region that is susceptible to proteolysis. It is suggested that the neck region assumes variable orientations by actin binding. In order to examine the effect of actin binding to α-actinin, we carried out limited digestion of α-actinin by chymotrypsin in the presence and absence of F-actin. Although the cleavage process was retarded when bound to F-actin, digestion to 32 kDa-head and 55 kDa-rod domains occurred through the same intermediate products as the digestion in the absence of F-actin. N-terminal sequencing of 55 kDa-fragment showed the neck region was cleaved at 276-Leu. The cleavage site was not affected by binding to F-actin nor ionic strength of the solvent. It was also indicated that α-actinin was cleaved at 15-Tyr by chymotrypsin. Quantitation of the cleavage products by densitometry of the SDS-gels suggested the conformational change of α-actinin at domain-connecting regions by F-actin binding.

No MeSH data available.