Biochemical Studies on the 10 nm Filaments of Avian Muscle

Author: Hubbard, Bruce D.

Year: 1979

Degree: Dissertation (Ph.D.)

Advisor: Lazarides, Elias

Committee Members: Lazarides, Elias; Revel, Jean-Paul; Owen, Ray David; Campbell, Judith L.; Hood, Leroy E.

Option: Biology

DOI: 10.7907/bq1f-ch51

Abstract

This thesis describes the purification and initial biochemical characterization of desmin, a major component of avian muscle 10 nm filaments.

1. Extraction of chicken gizzard with KI solubilizes most of the actomyosin and other fibrillar proteins, leaving behind a KI insoluble residue containing 10 nm filaments, desmin, and a small amount of insoluble actin. Antibodies prepared against electrophoretically purified desmin do not cross-react with other muscle proteins. In conjunction with indirect immunofluorescence, they demonstrate the presence of desmin in close association with the Z-lines and plasma membrane of skeletal muscle cells. Desmin also is associated with the Z-lines, intercalated discs, and intercellular adhesion sites of cardiac muscle cells.

2. Both desmin and the actin that remain in the KI extracted gizzard are insoluble in high salt, but are soluble at low pH or in agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant ratio of actin to desmin is attained.

3. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that actin is dissociated from the vast majority of desmin under these conditions. When the acetic acid is removed from actin-desmin solutions by dialysis against water, a gel forms that is composed of 12-14 nm diameter filaments. Both anti-actin and anti-desmin react uniformly with these filaments to a resolution limit of 250 nm.

4. Actin and desmin can be extracted from homogenized chicken gizzard in soluble form by 10 mM EGTA. Both proteins are retained on a DNase I affinity column and also coprecipitate in indirect immunoprecipitation with either anti-actin or anti-desmin. Velocity sedimentation of the EGTA extract shows that the actin and desmin cosediment as a series of stable complexes that range in size from 6 to 60 S. The 50-60 S family has an actin to desmin stoichiometry of approximately 5 to 1.

5. Several proteases are investigated that selectively cleave desmin (mwt 50,000) to sequentially produce fragments of 43,500 mwt, 40,000 mwt, and a 35,000 mwt limit digestion product that lacks 82% of the tyrosine of the original desmin. The cleavage process results in the conversion of gelled desmin to a non-cohesive, powdery, precipitate. These proteases remove the desmin from isolated myofibril bundles and cause their lateral separation into individual myofibrils.

6. Based upon these observations, it is proposed that desmin is a major component of muscle 10 nm filaments and that it forms stable complexes with actin. It is further proposed that the cellular function of desmin is to mechanically interlink individual actomyosin contractile units, both to each other and to the plasma membrane.

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