Reconstitution of Na+/K+-ATPase into phosphatidylcholine vesicles by dialysis of nonionic alkyl maltoside detergents.
The reconstitution of Na+/K+-ATPase from outer medulla of rabbit kidney into large unilamellar liposomes was achieved through detergent removal by dialysis of mixed micellar solutions of synthetic dioleoyl phosphatidylcholine/octyl glucoside and Na+/K+-ATPase/decyl maltoside or decenyl maltoside. Tight, transport-active liposomes were formed when the lipid and the enzyme were solubilized separately in the nonionic detergents and mixed immediately before starting the dialysis. The two maltoside detergents with different structures of the hydrophobic part of the molecule proved to be well suited for the solubilization of Na+/K+-ATPase with high retention of enzyme activity; the inactivation of enzyme being evidently slower with the unsaturated decenyl maltoside. The diameters of the proteoliposomes, 110 and 170 nm, respectively, were also dependent on the structure of the maltoside detergent, the saturated decyl maltoside producing the bigger liposomes. After freeze-fracture, both preparations exhibited intramembranous particles as structural indicators of successful reconstitution. The electrogenic activity of the reconstituted enzyme was determined by fluorescence measurements with Oxonol VI and by tracer-flux measurements with 22Na+.
Alpes
H
H
Department of Biology, University of Konstanz, F.R.G.
Apell
H J
HJ
Knoll
G
G
Plattner
H
H
Riek
R
R
D000818:Animals; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D003902:Detergents; D003956:Dialysis; D005614:Freeze Fracturing; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008854:Microscopy, Electron; D010713:Phosphatidylcholines; D011510:Proteolipids; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase; D013501:Surface-Active Agents
D003902:Detergents; D010713:Phosphatidylcholines; D011510:Proteolipids; D013501:Surface-Active Agents; C020485:proteoliposomes; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase