Asymmetric mass distribution of [formula omitted] in membranes studied by freeze-fracture-etch electron microscopy

Abstract

SDS-purified porcine kidney (Na + + K +)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: F A , which shows a rather smooth background with many intramembranous particles, and F B , which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that F A faces are covered with many intramembranous particles, while the etched external faces (E A) are either irregularly granulated or reveal many particles half the size of intramembranous particles. F B faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (E B) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated F A convex fracture faces and irregularly granulated or smooth etched E A surfaces, indicating that the F A face is the protoplasmic fracture face and that the majority of the protein mass of the (Na + + K +)-ATPase is located on the cytoplasmic half of the membrane.