Activation and inhibition of Na/K-ATPase by filipin-cholesterol complexation. A correlative biochemical and ultrastructural study on the microsomal and purified enzyme of the avian salt gland.
The Na/K-ATPase-rich microsomal fraction and purified Na/K-ATPase membranes of the salt-stressed avian salt gland were studied at defined filipin/cholesterol molar ratios (F/C) using enzyme assay and electron microscopy including negative staining, thin sectioning and freeze fracturing. Comparative examinations of detergent-treated microsomal fractions and the use of electron microscopic tracers revealed that F/C up to 2 activated latent Na/K-ATPase in sealed right-side-out vesicles by increasing membrane permeability without disrupting the vesicular membrane. Therefore, filipin offers an alternative to the detergents for the activation of latent vectorial membrane enzymes and a possible tool to examine their subcellular localization and sidedness in the membrane. The same F/C had no stimulatory effect on the microsomal anion-ATPase suggesting that the 2 ATPases are not located in the same membrane. Increasing F/C applied to the unfixed Na/K-ATPase membranes caused an increase in the number of structural F-C-complexes and a progressive lateral displacement of the enzyme particles which finally led to a separation of the areal distribution of these structures at F/C = 10. Such displacements did not occur in unfixed microsomes and were prevented by glutaraldehyde fixation of the purified membranes. F/C exceeding 2 progressively and temperature-dependently inhibited the Na/K-ATPase in its membrane-bound states, whereas the solubilized enzyme was rather insensitive. The structural and biochemical data suggest that inhibition results from the perturbation of the lipidic microenvironment of the enzyme caused by filipin-cholesterol complexation.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol / Q000494:pharmacology*; D004372:Ducks; D004789:Enzyme Activation; D005372:Filipin / Q000494:pharmacology*; D005614:Freeze Fracturing; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D011090:Polyenes / Q000494:pharmacology*; D012491:Salt Gland / Q000201:enzymology* / Q000648:ultrastructure; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*
D011090:Polyenes; D005372:Filipin; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase