Ouabain-binding vesicles from skeletal muscle.

The intact sacrospinalis (back) muscle of a rabbit was injected with ³H]ouabain and allowed to incubate for 20 min. The muscle was excised and homogenized and a microsomal preparation was made. This was placed on a sucrose density gradient and two protein bands were observed at 30 and 40% sucrose regions. Most of the ³H]ouabain was associated with the dense band. If the dense band was loaded with Ca oxalate and placed on a density gradient, then both protein and ouabain migrated equally to the bottom of the gradient. If the 40% band was treated with EDTA, both protein and ouabain migrated to 33% sucrose. It was therefore concluded that ouabain-binding vesicles were associated with the 40% band. If the 40% band was loaded with Ca oxalate and then passed through a French press and placed again on a gradient, the ouabain migrated to 22% sucrose while the protein bands appeared at 17 and 32% sucrose and in the pellet. It was concluded that ouabain-binding vesicles were distinct from the major vesicle population, but were mechanically linked until the linkage was destroyed by mechanical disruption. Electron microscopy revealed that the dense band of the gradient contained terminal cisternae and transverse tubules attached to the terminal cisternae in triad or diad junctions. It was concluded that the 40% band consisted mainly of terminal cisternae and that the ouabain-binding vesicles were transverse tubules attached to terminal cisternae. Transverse tubules therefore may be identified specifically after homogenization.