Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.
Open Access
- 1 September 1980
- journal article
- research article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 86 (3), 746-754
- https://doi.org/10.1083/jcb.86.3.746
Abstract
To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified [pig kidney] Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture EM after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 .ANG.. Also, freeze-fracture EM demonstrated uniform vesicles with diameters in the range of 700-1,100 .ANG. and an average value close to 900 .ANG.. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase, and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 .ANG. and similar to previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles/vesicle. Active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close to 90 .ANG. in the freeze-fracture micrographs.This publication has 29 references indexed in Scilit:
- Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.The Journal of cell biology, 1978
- Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPaseThe Journal of cell biology, 1977
- Evaluation of membrane structure facts and artefacts produced during freeze‐fracturingJournal of Microscopy, 1977
- A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medullaBiochimica et Biophysica Acta (BBA) - Biomembranes, 1977
- Ca++-induced fusion of proteoliposomes: Dependence on transmembrane osmotic gradientThe Journal of Membrane Biology, 1976
- Stereological method for estimating relative membrane surface area in freeze-fracture preparations of subcellular fractions*Journal of Microscopy, 1976
- Identification and transmembranous localization of active cytochrome oxidase in reconstituted membranes of purified phospholipids by electron microscopy.The Journal of cell biology, 1976
- Isolation and characterization of the components of the sodium pumpQuarterly Reviews of Biophysics, 1974
- The Fluid Mosaic Model of the Structure of Cell MembranesScience, 1972
- MEMBRANE SPLITTING IN FREEZE-ETCHINGThe Journal of cell biology, 1970