Purification of vacuolar ATPase with bafilomycin C1 affinity chromatography.
We have developed a fast and simple affinity purification method for vacuolar ATPases (V-ATPases) using bafilomycin C1 -coupled cellulose column. The purified protein from solubilized chicken medullary bone microsomal vesicles contains 8 subunits at sizes of 72, 65, 55, 41, 34, 20, 17 and 15 kDa. This subunit structure is typical to V-ATPases. Although the enzyme obtained is inactive, this method is useful in search of various isoforms of known V-ATPase subunits. We also present further evidence that the binding site of bafilomycin is located in the 17 kDa proteolipid subunit of V-ATPase.
Rautiala
T J
TJ
Department of Anatomy, University of Oulu, Finland.
Koskinen
A M
AM
Väänänen
H K
HK
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001665:Binding Sites; D001842:Bone and Bones / Q000201:enzymology*; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D018942:Macrolides; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology*; D008958:Models, Molecular; D008970:Molecular Weight; D010010:Osteoclasts / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D014961:X-Ray Diffraction
D000900:Anti-Bacterial Agents; D018942:Macrolides; D046911:Macromolecular Substances; C052502:bafilomycin C1; D006180:Proton-Translocating ATPases