title: Reconstitution of recombinant 40-kDa subunit of the clathrin-coated vesicle H(+)-ATPase.
abstract: We have proposed a model of the ATP hydrolytic sector of the clathrin-coated vesicle H(+)-ATPase wherein significant catalysis requires four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have cloned and expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity. This subunit lacks ATP hydrolytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subunit is required for vacuolar-type proton pump function.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8859.
authors.forename: S B
authors.initials: SB
authors.lastname: Peng
authors.forename: D K
authors.initials: DK
authors.lastname: Stone
authors.forename: X S
authors.initials: XS
authors.lastname: Xie
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002966:Clathrin; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D014617:Vacuoles / Q000201:enzymology
chemical_list: D002966:Clathrin; D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases