Isolation, characterization, and localization of the spanning protein from skeletal muscle triads.

Abstract

A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.