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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.


Cleavage map of skeletal muscle α-actinin. Cleavage sites of α-actinin by chymotrypsin were mapped on the primary sequence of skeletal muscle α-actinin (A). Flow chart for the degradation of 105 k-subunit into 55 k- and 32 k-domain (B). See the text for details.
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f4-7_29: Cleavage map of skeletal muscle α-actinin. Cleavage sites of α-actinin by chymotrypsin were mapped on the primary sequence of skeletal muscle α-actinin (A). Flow chart for the degradation of 105 k-subunit into 55 k- and 32 k-domain (B). See the text for details.

Mentions: Limited digestion with a protease is a useful method to access conformation of native proteins. α-Actinin is cleaved by chymotrypsin into protease resistant domains; namely N-terminal containing actin binding and rod-domain1. In a previous paper, we have shown that ionic strength of the solvents affects the conformation of skeletal muscle α-actinin molecule28. In the presence of 0.1 M KCl, the conformation of α-actinin is more compact than that in the absence of added salts. Reflecting the conformation, α-actinin becomes less susceptible to limited digestion by chymotrypsin although cleavage sites between domains were not affected (refer cleavage map shown in Fig. 4).


Limited digestion of α -actinin in the presence of F-actin
Cleavage map of skeletal muscle α-actinin. Cleavage sites of α-actinin by chymotrypsin were mapped on the primary sequence of skeletal muscle α-actinin (A). Flow chart for the degradation of 105 k-subunit into 55 k- and 32 k-domain (B). See the text for details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036780&req=5

f4-7_29: Cleavage map of skeletal muscle α-actinin. Cleavage sites of α-actinin by chymotrypsin were mapped on the primary sequence of skeletal muscle α-actinin (A). Flow chart for the degradation of 105 k-subunit into 55 k- and 32 k-domain (B). See the text for details.
Mentions: Limited digestion with a protease is a useful method to access conformation of native proteins. α-Actinin is cleaved by chymotrypsin into protease resistant domains; namely N-terminal containing actin binding and rod-domain1. In a previous paper, we have shown that ionic strength of the solvents affects the conformation of skeletal muscle α-actinin molecule28. In the presence of 0.1 M KCl, the conformation of α-actinin is more compact than that in the absence of added salts. Reflecting the conformation, α-actinin becomes less susceptible to limited digestion by chymotrypsin although cleavage sites between domains were not affected (refer cleavage map shown in Fig. 4).

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.