Ultrastructure of the purified and reconstituted Na/K-ATPase of the avian salt gland.
Na/K-ATPase of salt-stressed salt glands of the domestic duck (Anas platyrhynchos) was purified in membrane-bound form by incubation of the microsomal fraction with sodium dodecylsulphate and ATP followed by discontinuous sucrose gradient centrifugation. Gel electrophoresis of the purified plasma membrane preparation substantially showed the two polypeptide subunits of the Na/K-ATPase both of which stained with the periodic acid-Schiff reagent. About 99% of the total ATPase activity was ouabain-inhibitable amounting to 1300 mumol Pi/(mg protein X h) of specific activity. The anion-stimulated, ouabain-insensitive ATPase increased parallel to the Na/K-ATPase up to the microsomal fraction until it totally vanished during SDS incubation. Electron microscopy of thin sections revealed that the purified fraction consisted of flat and cup-shaped triple-layered membrane fragments. Particles arranged into clusters and strands were visible as 3 to 5 nm surface particles in negatively stained suspensions and as 8 to 10 nm intramembraneous particles in freeze fracture replicas. The differential distribution of the intramembraneous particles on the fracture faces reflected the structural membrane asymmetry. Solubilization of Na/K-ATPase led to the disappearance of intramembraneous particles. Incorporation of the solubilized enzyme into phosphatidylcholine vesicles again showed 8 to 10 nm particles apparently orientated at random in the artificial membrane. Control liposomes prepared in the absence of solubilized enzyme were devoid of intramembraneous particles. These results clearly demonstrate that the avian salt gland Na/K-ATPase exists as 8 to 10 nm particles in both the purified plasma membrane and the artificial phospholipid membrane.
D000818:Animals; D004372:Ducks / Q000378:metabolism*; D005614:Freeze Fracturing; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D008081:Liposomes; D008854:Microscopy, Electron; D012491:Salt Gland / Q000201:enzymology* / Q000648:ultrastructure; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*
D008081:Liposomes; D000254:Sodium-Potassium-Exchanging ATPase