ULTRACYTOCHEMICAL DEMONSTRATION OF FUNCTIONAL GROUPS IN THE LUMENAL PLASMA MEMBRANE OF RABBIT TRANSITIONAL EPITHELIUM

Abstract

The ultrathin sectioning, freeze-etching, negative staining, ruthenium red staining, ultracytochemical demonstration of sulfhydryl (SH) groups and performic acid-phosphotungustic acid (PFP) reaction were performed on intact and isolated lumenal plasma membranes of the normal rabbit urinary bladder. The lumenal membrane was characterized by a scalloped configuration with the concave plaque and crest-like interplaque regions. The concave plaque regions showed an asymmetric unit membrane of 12nm in width, having a thicker outer leaflet of 6nm which is composed of periodic dots. The plaque regions consisted of the hexagonal particle array (HPA) in the EF face and the interplaque regions smooth in the freeze-replica images. HPA, 13nm in diameter, was shown to be composed of 12 subparticles of 2.5nm in diameter in the negatively stained isolated membranes. The surface coat, 5-10nm in width, was demonstrated on the lumenal surface by a ruthenium red staining.The PFP reaction revealed the trilaminar structure in the interplaque regions, whereas the density (2nm in width) spanning membrane, resembling a ladder-like structure, was observed in the plaque regions. Hexagonally arranged electron lucent areas were also recognized in tangential sections. The substances stained with the PFP reaction may represent phospholipids and glycolipids in the lumenal plasma membrane. Reaction products for SH groups in the form of particles of 3-9nm in diameter were observed in and around the membrane in both the plaque and the interplaque regions and were often deposited directly on the lumenal membrane. It is suggested that the SH groups demonstrated here are derived from the surface proteins, perhaps enzyme proteins in the lumenal plasma membrane.