Vacuolar H(+)-ATPase of adrenal secretory granules. Rapid partial purification and reconstitution into proteoliposomes.
A procedure has been developed for the rapid purification and reconstitution into phospholipid vesicles of the proton-translocating ATPase of bovine adrenal chromaffin-granule membranes. It involves fractionation of the membranes with Triton X-114, resolubilization of the ATPase with n-octyl glucoside, addition of purified lipids and removal of detergent by gel filtration. The entire process can be completed within 2 h. H+ translocation was detected by the ATP-dependent quenching of the fluorescence of a permeant weak base. The effect of varying the lipid composition of the vesicles on ATP hydrolysis and H+ translocation by the reconstituted enzyme was examined. ATPase activity was maximally increased about 4-fold by added lipid, but was relatively insensitive to its composition, whereas vesicle acidification was absolutely dependent on the addition of phospholipids and cholesterol.
Perez-Castiñeira
J R
JR
Department of Biochemistry, University Medical School, Edinburgh, Scotland, U.K.
Apps
D K
DK
D000311:Adrenal Glands / Q000648:ultrastructure*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D008081:Liposomes / Q000378:metabolism*; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D011188:Potassium / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D011522:Protons; D013050:Spectrometry, Fluorescence; D014617:Vacuoles / Q000201:enzymology*; D014634:Valinomycin / Q000494:pharmacology
D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D011522:Protons; D014634:Valinomycin; D006180:Proton-Translocating ATPases; D011188:Potassium