title: Nucleotide labeling and reconstitution of the recombinant 58-kDa subunit of the vacuolar proton-translocating ATPase.
abstract: Evidence suggests that ATP hydrolysis catalyzed by the clathrin-coated vesicle proton-translocating ATPase requires at least four polypeptides of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D.K. (1988) J. Biol. Chem. 263, 9859-9867). To further investigate the subunit requirements for ATP hydrolysis, histidine-tagged, 58-kDa polypeptide was expressed in insect Sf9 (Spodoptera frugiperda) cells. After purification by Ni(2+)-nitrolotriacetic acid chromatography, the 58-kDa protein was found to lack significant ATPase activity. However, the subunit was photoaffinity labeled with [alpha-32P]ATP, [14C]ADP, or 35S-labeled ADP and UV irradiation in a divalent cation-dependent manner. The labeling was saturable with an apparent Kd of 4 microM for both ATP and ADP. ATP and ADP competition labeling experiments indicate that the two nucleotides share the same binding site. When reconstituted with recombinant 70-kDa subunit and a biochemically prepared catalytic sector (Vc) depleted of the 70- and 58-kDa subunits, the 58-kDa component restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.19 mumol Pi x mg protein-1 x min-1, thus demonstrating that ATP hydrolysis in vacuolar type proton pumps is dependent upon the 58-kDa subunit as well as multi-subunit interactions.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Peng
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002417:Cattle; D008969:Molecular Sequence Data; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases