Activation and partial purification of the ATPase of clathrin-coated vesicles and reconstitution of the proton pump.
A N-ethylmaleimide-sensitive ATPase was extracted and partially purified from clathrin-coated vesicles of bovine brain. During purification the enzyme lost activity which was restored by a purified phospholipid fraction from brain. Phosphatidylserine, but no other commercial phospholipids tested, replaced the brain lipid fraction as activator. Particles depleted of the ATPase exhibited no H+ pump activity when reconstituted with brain phospholipids by the cholate dilution procedure. H+ pump activity was restored by incubating the reconstituted vesicles with the partially purified ATPase.
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure; D002417:Cattle; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D002966:Clathrin; D010718:Phosphatidylserines; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide