Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain.
Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.
Xie
X S
XS
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235.
Stone
D K
DK
Racker
E
E
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure*; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008970:Molecular Weight; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology*
D000345:Affinity Labels; D014638:Vanadates; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide