Isolation of nebulin from rabbit skeletal muscle and its interaction with actin

Abstract

Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin, alpha-actinin, beta-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.

Figure 1

Purification of nebulin from rabbit skeletal muscle by DEAE-Toyopearl column chromatography. A muscle extract in 0.18 M KPO₄ (pH 7.0), 0.2 mM ATP, 0.1 mM MgCl₂, 0.1 mM EGTA, and 1 M urea was loaded to DEAE-Toyopearl column and linear urea gradient up to 4 M was applied. (A) SDS-PAGE patterns of each fraction of (B), (B) Elution pattern. a, applied sample; b, flow-through fraction; c–e, eluted fraction; f, overnight fraction with 4 M urea; HyAp, purified nebulin using hydroxylapatite column chromatography.

Figure 2

Immunoblot detection of purified nebulin. (a) SDS extract of whole rabbit back muscle, (b) nebulin fraction. (1) Amido black stained nitrocellulose sheets, (2) treated with PcNeb, (3) treated with Pc1200, (4) treated with Mc3B9, (5) treated with actin.

Figure 3

Modified purification of nebulin from rabbit skeletal muscle by DEAE-Toyopearl column chromatography. A muscle extract in 0.18 M KPO₄ (pH 7.0), 0.1 mM MgCl₂, 0.1 mM EGTA, and 1 M urea was loaded to DEAE-Toyopearl column and linear urea gradient up to 4 M was applied. (A) SDS-PAGE patterns of each fraction of (B), (B) Elution pattern. a, applied sample; b, flow-through fraction; c–e, eluted fraction; f, overnight fraction with 4 M urea.

Figure 4

Rotary shadowed images of purified nebulin from rabbit back muscle. (a) typical images of nebulin molecules. (b), enlarged images of arrow at (a). Bar, 200 nm.

Figure 5

Binding of purified nebulin and F-actin examined by cosedimentation. (a) nebulin only, (b) actin only, (c) nebulin + actin. (S) supernatant after centrifugation, (P) pellet after centrifugation. Nebulin and actin were mixed in 0.11 M KPO₄, 0.31 M urea, 0.27 mM ATP, and 0.1 mM MgCl₂, pH 7.0. Electrophoresis was performed on a 2–15% polyacrylamide gel.

Figure 6

Negative-stained electron micrographs of actin filaments under the influence of purification nebulin. (a) nebulin only, 19 μg/ml; (b) actin only, 0.2 mg/ml; (c and d) actin (0.2 mg/ml) was mixed with nebulin (19 μg/ml) in 0.11 M KPO₄, 0.31 M urea, 0.27 mM ATP and 0.1 mM MgCl₂, pH 7.0. Bar, 200 nm.

Figure 7

Binding of thin filament proteins to purified nebulin by far western blot analysis. Proteins (0.2 μg) adsorbed onto a nitrocellulose membrane were incubated in nebulin reaction solution (0.18 M KPO₄, 0.1 mM MgCl₂, 0.1 mM EGTA, and 0.5 M urea, pH 7.0) for 20 hours at 4°C. Amido black: amido black staining; +nebulin: incubation in nebulin (15 μg/ml), −nebulin: incubation in reaction solution alone.