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

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.

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Change in the amount of cleavage products detected during digestion. The amount of cleavage products were measured from digitized images of the SDS-gel with NIH ImageJ in the presence (A) and the absence (B) of F-actin. Ordinate, the relative amount of each product to the amount of 105 k-band in zero-time was expressed in percentage.
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f3-7_29: Change in the amount of cleavage products detected during digestion. The amount of cleavage products were measured from digitized images of the SDS-gel with NIH ImageJ in the presence (A) and the absence (B) of F-actin. Ordinate, the relative amount of each product to the amount of 105 k-band in zero-time was expressed in percentage.

Mentions: When bound to F-actin, the digestion of α-actinin was further retarded (Fig. 2B). In order to compare the amount of subfragments, we quantitated the amount of 105 k-, 87 k-, 72 k-, 68 k-, 55 k-, and 32 k-band from the recorded image of SDS-gels with NIH ImageJ. It was shown that 50% of the 105 kDa-subunit was digested after 10 hours of digestion in the presence of F-actin, while 85% was cleaved in the absence of actin (Fig. 3). Formation of 32 kDa-head and 55 kDa-rod domain were also retarded when α-actinin-F-actin complex was digested. Although F-actin affected the rate of the α-actinin digestion by chymotrypsin, the digestion to 55 kDa-rod and 32 kDa-head domains proceeded through common intermediate products with chain molecular weights of 98 k, 87 k, 72 k, and 68 k.


Limited digestion of α -actinin in the presence of F-actin
Change in the amount of cleavage products detected during digestion. The amount of cleavage products were measured from digitized images of the SDS-gel with NIH ImageJ in the presence (A) and the absence (B) of F-actin. Ordinate, the relative amount of each product to the amount of 105 k-band in zero-time was expressed in percentage.
© Copyright Policy
Related In: Results  -  Collection

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

f3-7_29: Change in the amount of cleavage products detected during digestion. The amount of cleavage products were measured from digitized images of the SDS-gel with NIH ImageJ in the presence (A) and the absence (B) of F-actin. Ordinate, the relative amount of each product to the amount of 105 k-band in zero-time was expressed in percentage.
Mentions: When bound to F-actin, the digestion of α-actinin was further retarded (Fig. 2B). In order to compare the amount of subfragments, we quantitated the amount of 105 k-, 87 k-, 72 k-, 68 k-, 55 k-, and 32 k-band from the recorded image of SDS-gels with NIH ImageJ. It was shown that 50% of the 105 kDa-subunit was digested after 10 hours of digestion in the presence of F-actin, while 85% was cleaved in the absence of actin (Fig. 3). Formation of 32 kDa-head and 55 kDa-rod domain were also retarded when α-actinin-F-actin complex was digested. Although F-actin affected the rate of the α-actinin digestion by chymotrypsin, the digestion to 55 kDa-rod and 32 kDa-head domains proceeded through common intermediate products with chain molecular weights of 98 k, 87 k, 72 k, and 68 k.

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.


Related in: MedlinePlus