Structural and chemical asymmetry of the calcium-transporting membranes of the sarcotubular system as revealed by electron microscopy

Abstract

The calcium-transporting membrane of the sarcotubular system appears as a triple-layered asymmetric structure with a total thickness of about 60 A after fixation with osmium tetroxide or with glutaraldehyde followed by osmium tetroxide. In the vesicles that are formed upon isolation of the membrane, the peripheral opaque membrane component is denser and thicker than the opaque component facing the interior of the vesicles. This pattern is also observed in the membranes of intact muscle. In experiments where the vesicles were incubated with the SH-reagent Hg-phenyl azoferritin, a close packing of ferritin particles at the outer surface of the vesicular membrane was observed. After treatment with NEM, which blocks the SH-groups, no ferritin was attached to the membranes and the vesicles were clustered together. When the SH-groups at the active sites which are specifically involved in the calcium transport were selectively protected with ATP before NEM treatment, the ferritin was packed at the outer membrane surface as in normal material. The results have been interpreted as indicating a preferred localization of the active sites involved in the calcium transport at the outer surface of the membrane. The distance between adjacent sites seems to correspond exactly to the resolution of the method using ferritin as SHlabeling reagent. In a second series of experiments vesicular preparations were sonicated or allowed to age as well as treated with distilled water or alkali solutions in order to expose the inner membrane surface to the ferritin compound. Also in these preparations the label was located almost exclusively at the outer surface of the membrane, indicating that fewer reactive sites are located at the inner than at the outer surface.