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Title: Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis

The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating {sup 125}I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from themore » amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis.« less
Authors:
; ;  [1]
  1. (Univ. of Pennsylvania School of Medicine, Philadelphia (USA))
Publication Date:
OSTI Identifier:
5264670
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; (USA); Journal Volume: 264:22
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIPS; ANTIGEN-ANTIBODY REACTIONS; BIOCHEMICAL REACTION KINETICS; CARBOHYDRATES; CELL MEMBRANES; CONCANAVALIN; CYTOPLASM; IODINE 125; MEMBRANE PROTEINS; MOLECULAR WEIGHT; MONOCLONAL ANTIBODIES; MUSCLES; PEPTIDE HYDROLASES; PROTEOLYSIS; RATS; RECEPTORS; TRACER TECHNIQUES; ANIMALS; ANTIBODIES; BETA DECAY RADIOISOTOPES; CELL CONSTITUENTS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; DECOMPOSITION; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; RADIOISOTOPES; REACTION KINETICS; RODENTS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques