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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.

No MeSH data available.


Time course of α-actinin digestion. α-Actinin was digested with 1/100 (w/w) chymotrypsin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at 25°C. (A) α-Actinin (0.42 mg/ml) was subjected to chymotrypitic digestion after clarification of α-actinin for 1 hr at 30,000 rpm, (B) α-Actinin-F-actin complex was pelleted by the ultra-centrifugation and dispersed in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at a protein concentration of 0.80 mg/ml. α-Actinin in the complex was digested with chymotrypsin. Numbers on the top of gel images indicate incubation time in hour. Numbers on the side of the image show molecular weight of marker proteins (MW) and those of cleavage products shown in k (×103). The 98 kDa-fragment that appeared only early period of digestion in small amount was marked by asterisk. The last lane in Plate B (-Fa5) showed the 5h-cleavage product without actin just shown for comparison of products. Polyacrylamide concentration, 10%. See the text for detail.
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f2-7_29: Time course of α-actinin digestion. α-Actinin was digested with 1/100 (w/w) chymotrypsin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at 25°C. (A) α-Actinin (0.42 mg/ml) was subjected to chymotrypitic digestion after clarification of α-actinin for 1 hr at 30,000 rpm, (B) α-Actinin-F-actin complex was pelleted by the ultra-centrifugation and dispersed in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at a protein concentration of 0.80 mg/ml. α-Actinin in the complex was digested with chymotrypsin. Numbers on the top of gel images indicate incubation time in hour. Numbers on the side of the image show molecular weight of marker proteins (MW) and those of cleavage products shown in k (×103). The 98 kDa-fragment that appeared only early period of digestion in small amount was marked by asterisk. The last lane in Plate B (-Fa5) showed the 5h-cleavage product without actin just shown for comparison of products. Polyacrylamide concentration, 10%. See the text for detail.

Mentions: In this report, we examined the cleavage pattern of α-actinin that bound to F-actin. α-Actinin (0.82 mg/ml) was mixed with 0.93 mg/ml of F-actin and centrifuged for 1 hr with a Hitachi 55P-72 ultracentrigue with a 40P rotor at 300,000 rpm. Pelleted α-actinin-F-actin complex was dispersed in 0.1 M KCl and 20 mM Tris-HCl, pH 7.5. Densitometry of the SDS-gel of the mixture indicated that the molar ratio of α-actinin to actin was 0.13, showing saturated binding of α-actinin to F-actin34,35. As F-actin was fairly resistant to chymotryptic digestion (refer Fig. 1B), concentration of α-actinin in the complex was estimated from the molar ratio and 1/100 (w/w) chymotrypsin (Sigma, type I-S) was added to the amount of α-actinin. Aliquot was withdrawn from the reaction mixture at reaction time shown in Figure 2 and the digestion was quenched with the SDS-solution. In control experiments, α-actinin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) was clarified by the centrifugation at 30,000 rpm and subjected to chymotrypsin cleavage. In the presence of 0.1 M KCl, the 105 kDa-subunit remained as a major component even after digestion over 10 hours with 1/100 (w/w) chymotrypsin (Fig. 1A). The 55 kDa-rod and 32 kDa-head domains were detected from one hour in digestion and their amounts increased gradually with time.


Limited digestion of α -actinin in the presence of F-actin
Time course of α-actinin digestion. α-Actinin was digested with 1/100 (w/w) chymotrypsin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at 25°C. (A) α-Actinin (0.42 mg/ml) was subjected to chymotrypitic digestion after clarification of α-actinin for 1 hr at 30,000 rpm, (B) α-Actinin-F-actin complex was pelleted by the ultra-centrifugation and dispersed in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at a protein concentration of 0.80 mg/ml. α-Actinin in the complex was digested with chymotrypsin. Numbers on the top of gel images indicate incubation time in hour. Numbers on the side of the image show molecular weight of marker proteins (MW) and those of cleavage products shown in k (×103). The 98 kDa-fragment that appeared only early period of digestion in small amount was marked by asterisk. The last lane in Plate B (-Fa5) showed the 5h-cleavage product without actin just shown for comparison of products. Polyacrylamide concentration, 10%. See the text for detail.
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f2-7_29: Time course of α-actinin digestion. α-Actinin was digested with 1/100 (w/w) chymotrypsin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at 25°C. (A) α-Actinin (0.42 mg/ml) was subjected to chymotrypitic digestion after clarification of α-actinin for 1 hr at 30,000 rpm, (B) α-Actinin-F-actin complex was pelleted by the ultra-centrifugation and dispersed in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) at a protein concentration of 0.80 mg/ml. α-Actinin in the complex was digested with chymotrypsin. Numbers on the top of gel images indicate incubation time in hour. Numbers on the side of the image show molecular weight of marker proteins (MW) and those of cleavage products shown in k (×103). The 98 kDa-fragment that appeared only early period of digestion in small amount was marked by asterisk. The last lane in Plate B (-Fa5) showed the 5h-cleavage product without actin just shown for comparison of products. Polyacrylamide concentration, 10%. See the text for detail.
Mentions: In this report, we examined the cleavage pattern of α-actinin that bound to F-actin. α-Actinin (0.82 mg/ml) was mixed with 0.93 mg/ml of F-actin and centrifuged for 1 hr with a Hitachi 55P-72 ultracentrigue with a 40P rotor at 300,000 rpm. Pelleted α-actinin-F-actin complex was dispersed in 0.1 M KCl and 20 mM Tris-HCl, pH 7.5. Densitometry of the SDS-gel of the mixture indicated that the molar ratio of α-actinin to actin was 0.13, showing saturated binding of α-actinin to F-actin34,35. As F-actin was fairly resistant to chymotryptic digestion (refer Fig. 1B), concentration of α-actinin in the complex was estimated from the molar ratio and 1/100 (w/w) chymotrypsin (Sigma, type I-S) was added to the amount of α-actinin. Aliquot was withdrawn from the reaction mixture at reaction time shown in Figure 2 and the digestion was quenched with the SDS-solution. In control experiments, α-actinin in 0.1 M KCl and 20 mM Tris-HCl (pH 7.5) was clarified by the centrifugation at 30,000 rpm and subjected to chymotrypsin cleavage. In the presence of 0.1 M KCl, the 105 kDa-subunit remained as a major component even after digestion over 10 hours with 1/100 (w/w) chymotrypsin (Fig. 1A). The 55 kDa-rod and 32 kDa-head domains were detected from one hour in digestion and their amounts increased gradually with time.

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.