title: Ca(2+) Binding and Transport Studied with Ca(2+)/EGTA Buffers and (45)Ca(2+).
abstract: The chapter describes procedures useful for determination of Ca(2+) binding by membranous Ca(2+)-ATPase based on the correction for the removal of Ca(2+) present in a non-bound state in the suspension medium. This is done by a filtration procedure that retains the membranous material on the Millipore filters. With suitable sucking devices it is possible to gently remove without dehydration nearly all medium from the Ca(2+) containing membranes, except that required for wetting of the filters on which they are deposited. Correction for this effect can be done with a double-filter where the radioactive content of the lower (protein-free) filter is subtracted from that present in the upper filter for calculation of Ca(2+) binding. This methodology can be used to study the effect of inhibitors on Ca(2+) binding and -transport, and with Ca(2+)/EGTA buffers to explore the Ca(2+) binding affinities and cooperative aspects of the two transport sites.
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark.|Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
authors.forename: Pankaj
authors.initials: P
authors.lastname: Sehgal
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.|Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Aarhus University, Aarhus C, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Olesen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark. jvm@biomed.au.dk.|Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Aarhus University, Aarhus C, Denmark. jvm@biomed.au.dk.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Møller
mesh_terms: D000818:Animals; D001692:Biological Transport; D002021:Buffers; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D002132:Calcium Radioisotopes; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004533:Egtazic Acid / Q000737:chemistry*; D011817:Rabbits
chemical_list: D002021:Buffers; D002132:Calcium Radioisotopes; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium
keywords: 45Ca2+; Calcium binding; Calcium transport; Filtration assay; SERCA