Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.

Abstract

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 ~) than the inner layer (20 ~). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (-70 •); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca ++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.

Morphology of isolated triads.

“…In our isolated triads, we find both by thin section and negative staining that the junctional feet are more tightly associated with the terminal cisternae than the transverse tubule, in keeping with previous observations by Franzini-Armstrong (4) and Campbell et al (31) by thin-section electron microscopy. Negative staining clearly shows that the junctional surface of the terminal cisternae is distinct from the remainder of the sarcoplasmic reticulum membrane since the surface appears to be devoid of the characteristic 30-40-/~ surface particles referable to calcium pump protein, the major component of the sarcoplasmic reticulum membrane (27). These conclusions are in keeping with the recent histochemical studies of Jorgensen et al (32).…”

“…Such feet structures have been previously observed, but only when tannic acid enhancement was used (26,27). The junction observed by thin section in isolated triads is similar in gap spacing and in ultrastructural detail to the feet observed in situ (Table I).…”

Effect of phospholipase A2 on purified gastric vesicles

“…The tannic acid method outlines the orientation of proteins in biologic membranes [14]. In intact tissue, distinct asymmetry of the plasma membrane is observed, indicating that penetration of tannic acid is not a limiting factor in this procedure.…”

Complex interactions between skeletal muscle ryanodine receptor and dihydropyridine receptor proteins

“…The first biochemical evidence for actual physical contact between the T-tubule and the terminal cisternae of the sarcoplasmic reticulum was the isolation of a fraction of intact triad junctions fkom a muscle homogenate (Caswell et al, 1976). The tannic acid mordant-staining technique was used to enhance the resolution of membrane structure (Saito et al, 1978) and to characterize structures protruding fkom the surface of isolated sarcoplasmic reticulum vesicles which were proposed to be the feet structures which span the 120 A gap between the T-tubule and the junctional terminal cisternae of sarcoplasmic reticulum (Campbell et al, 1980;Block et al, 1988).…”