title: Mutation of glutamate 309 to glutamine alters one Ca(2+)-binding site in the Ca(2+)-ATPase of sarcoplasmic reticulum expressed in Sf9 cells.
abstract: Sf9 cells infected with a baculovirus vector containing SERCA1 cDNA expressed immunoreactive rabbit fast-twitch muscle Ca(2+)-ATPase at levels up to 3 mg/liter. The microsomal fraction isolated from infected Sf9 cells catalyzed Ca2+ transport at rates 6-fold above control values. To obtain direct evidence for the postulate (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H., et al. (1989) Nature 339, 476-478) that Glu309 contributes to a Ca(2+)-binding site in the transmembrane sector of the Ca(2+)-ATPase, Ca2+ binding to wild type and mutant (Glu309 to Gln) Ca(2+)-ATPases was measured. The wild type Ca(2+)-ATPase, expressed in Sf9 cells and purified using a monoclonal antibody bound to Sepharose beads, bound approximately 1.6-1.7 mol Ca2+/mol of enzyme at 2 microM Ca2+ in a buffer favoring the E1 conformation of the enzyme and at 10 microM Ca2+ in a buffer favoring the E2 conformation. Under identical conditions, the mutant Ca(2+)-ATPase bound less than 0.1 mol of Ca2+/mol of enzyme in E1 buffer, but 0.8 mol Ca2+/mol in the E2 buffer. In spite of the ability of the Glu309 to Gln mutant enzyme to bind about 1 mol of Ca2+/mol of enzyme, E2P formation was not inhibited by up to 100 microM Ca2+, and E1P formation from ATP and Ca2+ was not observed with up to 100 microM Ca2+ in intact microsomal vesicles from Sf9 cells. Nevertheless, with detergent-solubilized and purified mutant Ca(2+)-ATPases, E2P formation was inhibited with a K0.5 of 2 microM Ca2+. These observations are consistent with the view that a single intact Ca(2+)-binding site is present in the mutant Ca(2+)-ATPase, which is accessible to Ca2+ only from the lumenal side and only in the E2 conformation. Transition from E2 to E1-Ca2+ may occur during or following Ca2+ binding, accounting for the relatively high Ca2+ affinity and inhibition by Ca2+ of phosphorylation from Pi.
authors.affiliation: Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
authors.forename: I S
authors.initials: IS
authors.lastname: Skerjanc
authors.forename: T
authors.initials: T
authors.lastname: Toyofuku
authors.forename: C
authors.initials: C
authors.lastname: Richardson
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium