title: Assay of Copper Transfer and Binding to P1B-ATPases.
abstract: P1B-type ATPases transport transition metals across biological membranes. The chemical characteristics of these substrates, as well as, physiological requirements have contributed to the evolution of high metal binding affinities (fM) in these enzymes. Metal binding determinations are consequently facilitated by the stable metal-protein interaction, while affinity measurements require careful analysis of metal levels. In the cell, transition metals are associated with chaperone proteins. Metals reach the ATPase transport sites following specific protein-protein interactions and ligand exchange enabling the metal transfer from the chaperone to the transporter. Here, we describe methods for analyzing the binding of Cu(+) to Cu(+)-ATPases, as well as the approach to monitor Cu(+) transfer from soluble Cu(+)-chaperones donors to and from membrane Cu(+)-ATPases.
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA.
authors.forename: Teresita
authors.initials: T
authors.lastname: Padilla-Benavides
authors.affiliation: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA, 01609, USA. arguello@wpi.edu.
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D001692:Biological Transport; D003300:Copper / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011485:Protein Binding; D011804:Quinolines / Q000737:chemistry; D012995:Solubility
chemical_list: D018832:Molecular Chaperones; D011804:Quinolines; D003300:Copper; D009532:Nickel; C047117:bicinchoninic acid; D000251:Adenosine Triphosphatases; D009571:Nitrilotriacetic Acid
keywords: Affinity; Binding; Copper; Cu+-ATPase s; Cu+-chaperones; P1B-type ATPases; Stoichiometry; Transition metals